Human Anti-IFN-gamma Neutralizing Antibodies as Selective IFN-gamma Pathway Inhibitors

ABSTRACT

This invention provides antibodies that interact with or bind to human interferon-gamma (IFN-γ) and methods for treating IFN-γ mediated diseases by administering a pharmaceutically effective amount of antibodies to IFN-γ. Methods of detecting the amount of IFN-γ in a sample using antibodies to IFN-γ are also provided.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of U.S. application Ser. No. 11/962,594,filed Dec. 21, 2007, which claims the benefit of the filing date of U.S.application Ser. No. 10/684,957, filed Oct. 14, 2003, now U.S. Pat. No.7,335,743, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 60/419,057, filed Oct. 16, 2002, and of U.S.Provisional Application No. 60/479,241, filed Jun. 17, 2003, thedisclosures of which are incorporated by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The invention relates to human monoclonal antibodies that bindinterferon gamma (IFN-γ). Compositions and methods for treating diseasesmediated by IFN-γ are also described.

BACKGROUND OF THE INVENTION

Interferons (IFNs) were originally named for their ability to interferewith viral infection of host cells (Isaacs and Lindenman, 1957, Proc. R.Soc. 147:28-267). Since their discovery, a number of members of theinterferon family have been identified with various biological roles inaddition to antiviral defense, including cell growth and cell immunity.Interferon types IFN-α, IFN-β, IFN-ω, and IFN-τ are type I interferonsand bind the type I IFN receptor, while IFN-γ is a type II interferonand binds the type II IFN receptor (Pfeffer et al., 1998, Cancer Res.58:2489-2499).

IFN-γ signaling depends on at least five distinct proteins: IFNGR1 andIFNGR2 (subunits of the IFN-γ receptor), Jak1, Jak2 and thetranscription factor STAT1 (Schindler and Darnell, 1995, Annu. Rev.Biochem. 64:621-651; Bach et al., 1997, Annu. Rev. Immunol. 15:563-591).IFN-γ receptors are found on most cell types, except mature erythrocytes(Farrar and Schreiber, 1993, Annu. Rev. Immunol. 11:571-611). Jak1,Jak2, and STAT1 proteins mediate IFN-γ signaling.

IFN-γ regulates a variety of biological functions, such as antiviralresponses, cell growth, immune response, and tumor suppression, andIFN-γ may mediate a variety of human diseases. Thus, there is a need inthe art for agents that can modulate the biological activity of IFN-γ.

SUMMARY OF THE INVENTION

The invention provides monoclonal antibodies that bind to interferon-γ(IFNγ) and polynucleotides that encode them. The antibodies may inhibitor modulate at least one of the biological activities of IFN-γ and canbe useful for ameliorating the effects of IFN-γ mediated diseases. Alsoprovided by the invention are hybridoma cells that produce and maysecrete into cell culture media the monoclonal antibodies of theinvention. The antibodies of the invention can be useful for treatingdiseases mediated by IFN-γ.

In certain aspects, the invention provides antibodies, optionallymonoclonal antibodies and/or human antibodies, which can comprise aheavy chain and a light chain, wherein the heavy chain comprises anamino acid sequence as set forth in SEQ ID NO: 2 or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, optionallymonoclonal antibodies, which may be human antibodies, comprising a heavychain and a light chain, wherein the heavy chain comprises an IgG1,IgG2, or an IgG4 heavy chain constant region. In some embodiments, anantibody of the invention comprises an amino acid sequence of the IgG1heavy chain constant region as set forth in SEQ ID NO: 2 or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In certain aspects, antibodies of the invention comprise a heavy chainand a light chain, wherein the variable region of the heavy chaincomprises an amino acid sequence as set forth in any of SEQ ID NO: 6,SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO: 30, or an antigen-binding oran immunologically functional immunoglobulin fragment thereof. In otheraspects, the light chain variable region comprises an amino acidsequence as set forth in any of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO:16, or SEQ ID NO: 31, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof. In additional aspects, theheavy chain comprises an amino acid sequence as set forth in any of SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 32, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof. In still further aspects, the light chain comprises an aminoacid sequence as set forth in any of SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, or SEQ ID NO: 33, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof.

The invention also provides antibodies that bind specifically to IFN-γ,wherein the heavy chain comprises a heavy chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 6, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, and the light chain comprises a light chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 8, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In certain aspects, the invention also provides antibodies andimmunologically functional immunoglobulin fragments thereof that canbind specifically to and/or inhibit or modulate the biological activityof IFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in SEQ ID NO: 6, and wherein thelight chain comprises a light chain variable region, and wherein thelight chain variable region comprises a sequence that has at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identityto the amino acid sequence as set forth in SEQ ID NO: 8, wherein theantibody interacts with IFN-γ.

The invention further provides antibodies that can inhibit or modulatethe biological activity of and/or specifically bind to IFN-γ, whereinthe heavy chain comprises a heavy chain variable region comprising anamino acid sequence as set forth in SEQ ID NO: 10, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof, andthe light chain comprises a light chain variable region comprising anamino acid sequence as set forth in SEQ ID NO: 12, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, which can inhibitor modulate the biological activity of and/or specifically bind toIFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in SEQ ID NO: 10, and wherein thelight chain comprises a light chain variable region, and wherein thelight chain variable region comprises a sequence that has at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identityto the amino acid sequence as set forth in SEQ ID NO: 12, wherein theantibody interacts with IFN-γ.

The invention further provides antibodies that can inhibit or modulatethe biological activity of and/or specifically bind to IFN-γ, whereinthe heavy chain comprises a heavy chain variable region comprising anamino acid sequence as set forth in SEQ ID NO: 30, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof, andthe light chain comprises a light chain variable region comprising anamino acid sequence as set forth in SEQ ID NO: 12, or an antigen-bindingor an immunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, which can inhibitor modulate the biological activity of and/or specifically bind toIFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in any of SEQ ID NO: 30, andwherein the light chain comprises a light chain variable region, andwherein the light chain variable region comprises a sequence that has atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about99% identity to the amino acid sequence as set forth in SEQ ID NO: 12,wherein the antibody interacts with IFN-γ.

The invention also provides antibodies that can inhibit or modulate thebiological activity of and/or bind specifically to IFN-γ, wherein theheavy chain comprises a heavy chain variable region comprising an aminoacid sequence as set forth in SEQ ID NO: 14, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and thelight chain comprises a light chain variable region comprising an aminoacid sequence as set forth in SEQ ID NO: 16, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, which canspecifically bind to and/or inhibit or modulate the biological activityof IFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in SEQ ID NO: 14, and wherein thelight chain comprises a light chain variable region, and wherein thelight chain variable region comprises an amino acid sequence that has atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about99% identity to the amino acid sequence as set forth in SEQ ID NO: 16,wherein the antibody interacts with IFN-γ.

The invention also provides antibodies that can inhibit or modulate thebiological activity of and/or bind specifically to IFN-γ, wherein theheavy chain comprises a heavy chain variable region comprising an aminoacid sequence as set forth in SEQ ID NO: 14, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and thelight chain comprises a light chain variable region comprising an aminoacid sequence as set forth in SEQ ID NO: 31, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, which can inhibitor modulate the biological activity of and/or specifically bind toIFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in SEQ ID NO: 14, and wherein thelight chain comprises a light chain variable region, and wherein thelight chain variable region comprises an amino acid sequence that has atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about99% identity to the amino acid sequence as set forth in SEQ ID NO: 31,wherein the antibody interacts with IFN-γ.

The invention also provides antibodies that can inhibit or modulate thebiological activity of and/or bind specifically to IFN-γ, wherein theheavy chain comprises an amino acid sequence as set forth in SEQ ID NO:17 or an antigen-binding or an immunologically functional immunoglobulinfragment thereof, and the light chain comprises an amino acid sequenceas set forth in SEQ ID NO: 18, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, which can inhibitor modulate the biological activity of and/or bind specifically toIFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in SEQ ID NO: 17, and wherein thelight chain comprises a light chain variable region, and wherein thelight chain variable region comprises an amino acid sequence that has atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about99% identity to the amino acid sequence as set forth in SEQ ID NO: 18,wherein the antibody interacts with IFN-γ.

The invention further provides antibodies that can inhibit or modulatethe biological activity of and/or bind specifically to IFN-γ, whereinthe heavy chain comprises an amino acid sequence as set forth in SEQ IDNO: 19, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises an aminoacid sequence as set forth in SEQ ID NO: 20, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, which can inhibitor modulate the biological activity of and/or bind specifically toIFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in any of SEQ ID NO: 19, andwherein the light chain comprises a light chain variable region, andwherein the light chain variable region comprises an amino acid sequencethat has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or about 99% identity to the amino acid sequence as set forth in SEQ IDNO: 20, wherein the antibody interacts with IFN-γ.

The invention also provides antibodies that can inhibit or modulate thebiological activity of and/or bind specifically to IFN-γ, wherein theheavy chain comprises an amino acid sequence as set forth in SEQ ID NO:21, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises an aminoacid sequence as set forth in SEQ ID NO: 22, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, which can inhibitor modulate the biological activity of and/or bind specifically toIFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in any of SEQ ID NO: 21, andwherein the light chain comprises a light chain variable region, andwherein the light chain variable region comprises an amino acid sequencethat has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or about 99% identity to the amino acid sequence as set forth in SEQ IDNO: 22, wherein the antibody interacts with IFN-γ.

The invention also provides antibodies that can inhibit or modulate thebiological activity of and/or bind specifically to IFN-γ, wherein theheavy chain comprises an amino acid sequence as set forth in SEQ ID NO:32, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises an aminoacid sequence as set forth in SEQ ID NO: 20, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, which can inhibitor modulate the biological activity of and/or bind specifically toIFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in SEQ ID NO: 32, and wherein thelight chain comprises a light chain variable region, and wherein thelight chain variable region comprises an amino acid sequence that has atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about99% identity to the amino acid sequence as set forth in SEQ ID NO: 20,wherein the antibody interacts with IFN-γ.

The invention also provides antibodies that can inhibit or modulate thebiological activity of and/or bind specifically to IFN-γ, wherein theheavy chain comprises an amino acid sequence as set forth in SEQ ID NO:21, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof, and the light chain comprises an aminoacid sequence as set forth in SEQ ID NO: 33, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies, which can inhibitor modulate the biological activity of and/or bind specifically toIFN-γ, comprising a heavy chain and a light chain, wherein the heavychain comprises a heavy chain variable region, and wherein the heavychain variable region comprises a sequence that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identity tothe amino acid sequence as set forth in SEQ ID NO: 21, and wherein thelight chain comprises a light chain variable region, and wherein thelight chain variable region comprises an amino acid sequence that has atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about99% identity to the amino acid sequence as set forth in SEQ ID NO: 33,wherein the antibody interacts with IFN-γ.

The invention also provides single chain antibodies, single chain Fvantibodies, F(ab) antibodies, F(ab)′ antibodies and (Fab)₂ antibodies.

In particular aspects, the invention provides a light chain comprisingan amino acid sequence as set forth in any of SEQ ID NO: 8, SEQ ID NO:12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ IDNO: 31, or SEQ ID NO: 33 or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof.

In addition, the invention provides a heavy chain comprising an aminoacid sequence as set forth in any of SEQ ID NO: 6, SEQ ID NO: 10, SEQ IDNO: 14, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 30, orSEQ ID NO: 32, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

The invention also relates to isolated human antibodies thatspecifically bind IFN-γ, wherein the antibody comprises: (a) human heavychain framework regions, a human heavy chain CDR1 region, a human heavychain CDR2 region, and a human heavy chain CDR3 region; and (b) humanlight chain framework regions, a human light chain CDR1 region, a humanlight chain CDR2 region, and a human light chain CDR3 region. In certainaspects, the human heavy chain CDR1 region can be the heavy chain CDR1region of monoclonal antibodies (mAbs) 1119, 1118, 1118*, or 1121 asshown in FIG. 12 and SEQ ID NO:34 and the human light chain CDR1 regioncan be the light chain CDR1 region of mAbs 1119, 1118, 1121*, or 1121 asshown in FIG. 13 and SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40. Inother aspects, the human heavy chain CDR2 region can be the heavy chainCDR2 region of mAbs 1119, 1118, 1118*, or 1121 as shown in FIG. 12 andSEQ ID NO:35 and the human light chain CDR2 region can be the lightchain CDR2 region of mAbs 1119, 1118, 1121*, or 1121 as shown in FIG. 13and SEQ ID NO:41 or SEQ ID NO:42. In still other aspects, the humanheavy chain CDR3 region is the heavy chain CDR3 region of mAbs 1119,1118, 1118*, or 1121 as shown in FIG. 12 and SEQ ID NO:36 or SEQ IDNO:37, and the human light chain CDR3 region is the light chain CDR3region of mAbs 1119, 1118, 1121*, or 1121 as shown in FIG. 13 and SEQ IDNO:43 or SEQ ID NO:44.

In addition, the invention provides methods for treating a diseaseassociated with increased production of or sensitivity to IFN-γ and/or adisease mediated by IFN-γ comprising the step of administering apharmaceutically effective amount of one or a plurality of monoclonalantibodies of the invention or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof to an individual in needthereof.

The invention also provides methods for detecting the level of IFN-γ ina biological sample, comprising the step of contacting the sample with amonoclonal antibody of the invention or antigen-binding fragmentthereof.

The invention also provides an isolated antibody that can bindspecifically to and/or inhibit or modulate the biological activity ofIFN-γ and comprises a heavy chain CDR3 having an amino acid sequencethat is: (a) an amino acid sequence consisting of at least 7 of theamino acids of SEQ ID NO:36 in the same order and spacing as they occurin SEQ ID NO:36; or (b) an amino acid sequence comprising SEQ ID NO:37.The antibody can further comprise a light chain CDR3 having an aminoacid sequence that is: (a) an amino acid sequence consisting at least 8of the amino acids of SEQ ID NO:43 in the same order and spacing as theyare in SEQ ID NO:43; or (b) an amino acid sequence consisting of atleast 9 of the amino acids of SEQ ID NO:44 in the same order and spacingas they are in SEQ ID NO:44. The antibody can further comprise one ormore CDR selected from the group consisting of: SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQID NO:42. The heavy chain CDR3 may consist of at least the amino acidsof SEQ ID NO:36 and the light chain CDR3 may consist of at least theamino acids of SEQ ID NO:43.

In addition, the invention provides isolated antibodies that can bindspecifically to and/or inhibit or modulate the biological activity ofIFN-γ comprising a light chain CDR3 having an amino acid sequence thatis: (a) an amino acid sequence consisting of at least 8 of the aminoacids of SEQ ID NO:43 in the same order and spacing as they occur in SEQID NO:43; or (b) an amino acid sequence consisting of at least 9 of theamino acids of SEQ ID NO:44 in the same order and spacing as they occurin SEQ ID NO:44. The antibody can further comprise a CDR having theamino acid sequence of SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, or SEQ ID NO:42.

An isolated antibody of the invention, which can specifically bind toand/or inhibit or modulate the biological activity of IFN-γ, cancomprise six CDRs having at least the amino acid sequences of: (a) SEQID NO:34; (b) SEQ ID NO:35; (c) SEQ ID NO:36 or SEQ ID NO:37; (d) SEQ IDNO:38, SEQ ID NO:39, or SEQ ID NO:40; (e) SEQ ID NO:41 or SEQ ID NO:42;and (f) SEQ ID NO:43 or SEQ ID NO:44.

Isolated antibodies of the invention, which can specifically bind toand/or inhibit or modulate the biological activity of IFN-γ, cancomprise an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or about 99% identical to SEQ ID NO:6, SEQ IDNO:10, SEQ ID NO:14, or SEQ ID NO:30, wherein the alignment between theamino acid sequence and SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, or SEQID NO:30 spans at least 50 amino acids, and/or an amino acid sequence atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about99% identical to SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ IDNO:31, wherein the alignment between the amino acid sequence and SEQ IDNO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:31 spans at least 50amino acids.

In another aspect, antibodies of the invention, which can specificallybind to and/or inhibit or modulate the biological activity of IFN-y, cancomprise an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or about 99% identical to SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, or SEQ ID NO:32, wherein the alignment between theamino acid sequence and SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQID NO:32 spans at least 50 amino acids, and/or an amino acid sequence atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about99% identical to SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ IDNO:33, wherein the alignment between the amino acid sequence and SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:33 spans at least 50amino acids. One amino acid sequence within these antibodies cancomprise at least 5 of the amino acids in SEQ ID NO:36 or SEQ ID NO:37,in the same order and spacing as they occur in SEQ ID NO:36 or SEQ IDNO:37, and/or one amino acid sequence within these antibodies cancomprise at least 6 of the amino acids in SEQ ID NO:43 or SEQ ID NO:44in the same order and spacing as they occur in SEQ ID NO:43 or SEQ IDNO:44.

In one aspect, the invention provides an antibody, which can be anisolated fully human antibody, wherein the antibody can inhibit ormodulate the biological activity of human IFN-γ. In another aspect, anantibody of the invention, which can be an isolated fully humanantibody, cannot inhibit or modulate the biological activity ofcynomolgus monkey and murine IFN-γ. In yet another aspect, a fully humanantibody of the invention can inhibit or modulate the biologicalactivity of human and chimpanzee IFN-γ, but cannot inhibit or modulatethe biological activity of cynomolgus monkey and/or murine IFN-γ. Instill another aspect, substitution of residue 19 of human IFN-γ withaspartic acid and/or residue 20 with proline prevents or antagonizes theinhibition of the biological activity of human IFN-γ by the antibody.Further, the antibody can inhibit the biological activity of a mutatedversion of cynolmolgus monkey IFN-γ substituted at residues 19, 20, and65 with histidine, serine, and serine, respectively.

In yet further aspects, isolated antibodies of the invention, which canbind specifically to and/or inhibit or modulate the biological activityof IFN-γ, can comprise an amino acid sequence at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:32, wherein thealignment between the amino acid sequence and SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, or SEQ ID NO:32 spans at least 50 amino acids,and/or can comprise an amino acid sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:33, wherein thealignment between the amino acid sequence and SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, or SEQ ID NO:33 spans at least 50 amino acids.

In a further embodiment, the invention encompasses an isolated antibodythat can bind specifically to IFN-γ comprising (a) an amino acidsequence comprising SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, or SEQ IDNO:30, or a fragment of one of these sequences and (b) an amino acidsequence comprising SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ IDNO:31, or a fragment of one of these sequences. The antibody maycomprise a heavy chain and a light chain. The antibody may comprise (a)SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:30 and (b) SEQ IDNO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:31.

Further, any of the antibodies of the invention can be humanizedantibodies or fully human antibodies.

The invention also provides polynucleotides, including isolatedpolynucleotides, that encode any of the antibodies of the invention orportions thereof as described herein, including CDR regions, heavy chainvariable regions, light chain variable regions, single chain antibodies,single chain Fv antibodies, F(ab) antibodies, F(ab)′ antibodies and(Fab′)₂ antibodies. The invention further provides vectors comprisingsuch polynucleotides and host cells, optionally mammalian host cells,containing such polynucleotides and/or vectors. Antibodies of theinvention can be produced by culturing such host cells.

Specific preferred embodiments of the invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict a nucleotide sequence of a portion of a cDNA (FIG.1A; SEQ ID NO:1) encoding an amino acid sequence (FIG. 1B; SEQ ID NO:2)of a heavy chain constant region of the 1118, 1118*, 1119, 1121, and1121* anti-IFN-γ antibodies.

FIGS. 2A-2B depict the nucleotide sequence of a portion of a cDNA (FIG.2A; SEQ ID NO:3) encoding an amino acid sequence (FIG. 2B; SEQ ID NO:4)of a light chain constant region of the 1118, 1118*, 1119, 1121, and1121* anti-IFN-γ antibodies.

FIGS. 3A-3B depict the nucleotide sequence of a portion of a cDNA (FIG.3A; SEQ ID NO:5) encoding an amino acid sequence (FIG. 3B; SEQ ID NO:6)of the heavy chain variable region of the 1119 anti-IFN-γ antibody.

FIGS. 4A-4B depict the nucleotide sequence of a portion of a cDNA (FIG.4A; SEQ ID NO:7) encoding an amino acid sequence (FIG. 4B; SEQ ID NO: 8)of the light chain variable region of the 1119 anti-IFN-γ antibody.

FIG. 5A-5B depict the nucleotide sequence of a portion of a cDNA (FIG.5A; SEQ ID NO: 9) encoding the amino acid sequence (FIG. 5B; SEQ IDNO:10) of the heavy chain variable region of the 1118 anti-IFN-γantibody. FIG. 5C depicts the amino acid sequence of the heavy chainvariable region (SEQ ID NO:30) of the 1118* anti-IFN-γ antibody. FIG. 5Ddepicts the nucleotide sequence of the heavy chain variable region (SEQID NO:56) of the 1118* anti-IFN-γ antibody.

FIGS. 6A-6B depict the nucleotide sequence of a portion of a cDNA (FIG.6A; SEQ ID NO:11) encoding the amino acid sequence (FIG. 6B; SEQ IDNO:12) of the light chain variable region of the 1118 or 1118*anti-IFN-γ antibody.

FIGS. 7A-7B depict the nucleotide sequence of a portion of a cDNA (FIG.7A; SEQ ID NO:13) encoding the amino acid sequence (FIG. 7B; SEQ IDNO:14) of the heavy chain variable region of the 1121 or 1121*anti-IFN-γ antibody.

FIG. 8A-B depict the nucleotide sequence of a portion of a cDNA (FIG.8A; SEQ ID NO:15) encoding the amino acid sequence (FIG. 8B; SEQ IDNO:16) of the light chain variable region of the 1121 anti-IFN-γantibody. FIG. 8C depicts the amino acid sequence (SEQ ID NO:31) of thelight chain variable region of the 1121* anti-IFN-γ antibody. FIG. 8Ddepicts the nucleotide sequence (SEQ ID NO:57) of the light chainvariable region of the 1121* anti-IFN-γ antibody.

FIG. 9 contains a graph showing neutralization or inhibition of thebiological activity of IFN-γ in the A549 bioassay with the 1119, 1118,and 1121 monoclonal antibodies.

FIG. 10 contains a graph showing neutralization or inhibition of thebiological activity of IFN-γ in the THP-1/HLA DR bioassay by the 1119,1118, and 1121 monoclonal antibodies.

FIG. 11 contains a graph showing neutralization or inhibition of thebiological activity of IFN-γ in a whole blood bioassay (two donors) bythe 1119 monoclonal antibody.

FIG. 12 shows an alignment of an amino terminal portion (including thevariable region) of the heavy chains of the anti-IFN-γ monoclonalantibodies designated 1118, 1118*, 1121, and 1119. The sequences includethe signal sequence encoded on the cDNAs isolated in Example 3. Thesignal sequence extends from position 1 through 19. CDRs are underlined.As depicted in the Figure, CDR1 spans from amino acid 50-54, CDR2 spansfrom 69-85, and CDR3 spans from 118-125. The numbering system of Kabatet al. (1991, Sequences of Proteins of Immunological Interest, PublicHealth Service N.I.H., Bethesda, Md.) starts at the first amino acid ofthe mature antibody and excludes the signal sequence. Thus, position 20in this Figure would correspond to position 1 of Kabat et al. (supra).

FIG. 13 shows an alignment of an amino terminal portion (including thevariable region) of the light chains of the anti-IFN-γ monoclonalantibodies designated 1118, 1121, 1121*, and 1119. The sequences includethe signal sequence encoded on the cDNAs isolated in Example 3. Thesignal sequence extends from position 1 through 20. CDRs are underlined.As depicted in the Figure, CDR1 spans from amino acid 44-55, CDR2 spansfrom 71-77, and CDR3 spans from 110-118. Since the numbering system ofKabat et al. (supra) excludes the signal sequence, position 21 in thisFigure corresponds to position 1 of Kabat et al.

FIG. 14 shows production of IP-10 in response to IFN-γ by whole bloodtaken from a chimpanzee at 2 or 1 week(s) prior to (lines labeled “−2”and “−1” in FIG. 14) or 2, 8, 15, 29, or 36 days after (lines labeled“2,” “8,” “15,” “29,” or “36” in FIG. 14) the start of a course of 3injections of anti-IFN-γ antibody, which occurred once per week.

FIG. 15 is similar to FIG. 14 except that the blood of a differentchimpanzee was used.

DETAILED DESCRIPTION OF THE INVENTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited in this application are expressly incorporated byreference herein for any purpose.

DEFINITIONS

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al., 2001, MOLECULARCLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., which is incorporated herein byreference for any purpose. Unless specific definitions are provided, thenomenclature utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques may be usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the isolated polynucleotide (1)is not associated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, (2) is linked to apolynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldtypically be found in nature, (2) is essentially free of other proteinsfrom the same source, e.g., from the same species, (3) is expressed by acell from a different species, (4) has been separated from at leastabout 50 percent of polynucleotides, lipids, carbohydrates, or othermaterials with which it is associated in nature, (5) is not associated(by covalent or noncovalent interaction) with portions of a protein withwhich the “isolated protein” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated protein can be encoded by genomic DNA, cDNA,mRNA or other RNA, of synthetic origin, or any combination thereof.Preferably, the isolated protein is substantially free from proteins orpolypeptides or other contaminants that are found in its naturalenvironment that would interfere with its use (therapeutic, diagnostic,prophylactic, research or otherwise).

The terms “polypeptide” or “protein” means one or more chains of aminoacids, wherein each chain comprises amino acids covalently linked bypeptide bonds, and wherein said polypeptide or protein can comprise aplurality of chains non-covalently and/or covalently linked together bypeptide bonds, having the sequence of native proteins, that is, proteinsproduced by naturally-occurring and specifically non-recombinant cells,or genetically-engineered or recombinant cells, and comprise moleculeshaving the amino acid sequence of the native protein, or moleculeshaving deletions from, additions to, and/or substitutions of one or moreamino acids of the native sequence. The terms “polypeptide” and“protein” specifically encompass anti-IFN-γ antibodies, or sequencesthat have deletions from, additions to, and/or substitutions of one ormore amino acid of an anti-IFN-γ antibody. Thus, a “polypeptide” or a“protein” can comprising one (termed “a monomer”) or a plurality (termed“a multimer”) of amino acid chains.

The term “polypeptide fragment” refers to a polypeptide, which can bemonomeric or multimeric, that has an amino-terminal deletion, acarboxyl-terminal deletion, and/or an internal deletion or substitutionof a naturally-occurring or recombinantly-produced polypeptide. Incertain embodiments, a polypeptide fragment can comprise an amino acidchain at least 5 to about 500 amino acids long. It will be appreciatedthat in certain embodiments, fragments are at least 5, 6, 8, 10, 14, 20,50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long.Particularly useful polypeptide fragments include functional domains,including binding domains. In the case of an anti-IFN-γ antibody, usefulfragments include, but are not limited to: a CDR region, especially aCDR3 region of the heavy or light chain; a variable domain of a heavy orlight chain; a portion of an antibody chain or just its variable regionincluding two CDRs; and the like.

The term “immunologically functional immunoglobulin fragment” as usedherein refers to a polypeptide fragment that contains at least the CDRsof the immunoglobulin heavy and light chains. An immunologicallyfunctional immunoglobulin fragment of the invention is capable ofbinding to an antigen. In preferred embodiments, the antigen is a ligandthat specifically binds to a receptor. In these embodiments, binding ofan immunologically functional immunoglobulin fragment of the inventionprevents or inhibits binding of the ligand to its receptor, interruptingthe biological response resulting from ligand binding to the receptor.Preferably, an immunologically functional immunoglobulin fragment of theinvention binds specifically to IFN-γ. Most preferably, the fragmentbinds specifically to and/or inhibits or modulates the biologicalactivity of human IFN-γ.

The term “naturally-occurring” as used herein and applied to an objectrefers to the fact that the object can be found in nature. For example,a polypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andthat has not been intentionally modified by man is naturally-occurring.

The term “operably linked” means that the components to which the termis applied are in a relationship that allows them to carry out theirinherent functions under suitable conditions. For example, atranscription control sequence “operably linked” to a protein codingsequence is ligated thereto so that expression of the protein codingsequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “control sequence” as used herein refers to polynucleotidesequences that can affect expression, processing or intracellularlocalization of coding sequences to which they are ligated. The natureof such control sequences may depend upon the host organism. Inparticular embodiments, transcription control sequences for prokaryotesmay include a promoter, ribosomal binding site, and transcriptiontermination sequence. In other particular embodiments, transcriptioncontrol sequences for eukaryotes may include promoters comprising one ora plurality of recognition sites for transcription factors,transcription enhancer sequences, transcription termination sequencesand polyadenylation sequences. In certain embodiments, “controlsequences” can include leader sequences and/or fusion partner sequences.

The term “polynucleotide” as referred to herein means single-stranded ordouble-stranded nucleic acid polymers of at least 10 bases in length. Incertain embodiments, the nucleotides comprising the polynucleotide canbe ribonucleotides or deoxyribonucleotides or a modified form of eithertype of nucleotide. Said modifications include base modifications suchas bromuridine, ribose modifications such as arabinoside and2′,3′-dideoxyribose and internucleotide linkage modifications such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate andphosphoroamidate. The term “polynucleotide” specifically includes singleand double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and/or non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset comprising members that aregenerally single-stranded and have a length of 200 bases or fewer. Incertain embodiments, oligonucleotides are 10 to 60 bases in length. Incertain embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18,19, or 20 to 40 bases in length. Oligonucleotides may be single strandedor double stranded, e.g. for use in the construction of a gene mutant.Oligonucleotides of the invention may be sense or antisenseoligonucleotides with reference to a protein-coding sequence.

Unless specified otherwise, the left-hand end of single-strandedpolynucleotide sequences is the 5′ end; the left-hand direction ofdouble-stranded polynucleotide sequences is referred to as the 5′direction. The direction of 5′ to 3′ addition of nascent RNA transcriptsis referred to as the transcription direction; sequence regions on theDNA strand having the same sequence as the RNA and which are 5′ to the5′ end of the RNA transcript are referred to as “upstream sequences”;sequence regions on the DNA strand having the same sequence as the RNAand which are 3′ to the 3′ end of the RNA transcript are referred to as“downstream sequences”.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” includesnucleotides with modified or substituted sugar groups and the like. Theterm “oligonucleotide linkages” includes oligonucleotide linkages suchas phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl.Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077;Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991,Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES ANDANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), OxfordUniversity Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510;Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures ofwhich are hereby incorporated by reference for any purpose. Anoligonucleotide can include a detectable label to enable detection ofthe oligonucleotide or hybridization thereof.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.

The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control expression of inserted heterologous nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and RNA splicing, if introns are present.

The term “host cell” is used to refer to a cell into which has beenintroduced, or is capable of being introduced with a nucleic acidsequence and further expresses or is capable of expressing a selectedgene of interest. The term includes the progeny of the parent cell,whether or not the progeny is identical in morphology or in geneticmake-up to the original parent, so long as the selected gene is present.

The term “transduction” is used to refer to the transfer of genes fromone bacterium to another, usually by a phage. “Transduction” also refersto the acquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook etal., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring HarborLaboratories; Davis et al., 1986, BASIC METHODS 1N MOLECULAR BIOLOGY,Elsevier; and Chu et al., 1981, Gene 13:197. Such techniques can be usedto introduce one or more exogenous DNA moieties into suitable hostcells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, or may be maintained transiently as an episomal element withoutbeing replicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. An epitope is a region of an antigen that is bound by anantibody. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments,may have specific three-dimensional structural characteristics, and/orspecific charge characteristics. In certain embodiments, an antibody issaid to specifically bind an antigen when it preferentially recognizesits target antigen in a complex mixture of proteins and/ormacromolecules. An antibody is said to specifically bind an antigen whenthe equilibrium dissociation constant is ≦10⁻⁷ or 10⁻⁸ M. In someembodiments, the equilibrium dissociation constant may be ≦10⁻⁹ M or≦10⁻¹⁰ M.

As used herein, when a first sequence consists of, for example, 10 aminoacids of the sequence RASQSVSSSY (SEQ ID NO: 56), another sequence has 7amino acids in the “same order and spacing” as they occur in the firstsequence if 7 amino acids are identical to those in the sequence andoccur in the same relative positions as they occur in the sequence. Forexample, a sequence RAAAAVSSSY (SEQ ID NO: 57) has 7 amino acids in thesame order and spacing as they occur in RASQSVSSSY (SEQ ID NO: 56). Incontrast, this is not true for a sequence RASSVSSSY (SEQ ID NO: 58),since it contains an internal deletion relative to RASQSVSSSY (SEQ IDNO: 56), with 3 and 6 amino acids on either side of the deletion.Therefore, it has at most 6 amino acids in the same order and spacing asthe first sequence. The shortest possible sequence that could have 7amino acids in the same order and spacing as in RASQSVSSSY (SEQ ID NO:56) would be 7 amino acids long, for example SQSVSSS (SEQ ID NO: 59).

The term “identity,” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequencesthereof. In the art, “identity” also means the degree of sequencerelatedness between nucleic acid molecules or polypeptides, as the casemay be, as determined by the match between strings of two or morenucleotide or two or more amino acid sequences. “Identity” measures thepercent of identical matches between the smaller of two or moresequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”).

The term “similarity” is used in the art with regard to a relatedconcept, but in contrast to “identity,” “similarity” refers to a measureof relatedness, which includes both identical matches and conservativesubstitution matches. If two polypeptide sequences have, for example,10/20 identical amino acids, and the remainder are all non-conservativesubstitutions, then the percent identity and similarity would both be50%. If in the same example, there are five more positions where thereare conservative substitutions, then the percent identity remains 50%,but the percent similarity would be 75% (15/20). Therefore, in caseswhere there are conservative substitutions, the percent similaritybetween two polypeptides will be higher than the percent identitybetween those two polypeptides.

Identity and similarity of related nucleic acids and polypeptides can bereadily calculated by known methods. Such methods include, but are notlimited to, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk,A. M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING:INFORMATICS AND GENOME PROJECTS, (Smith, D. W., ed.), 1993, AcademicPress, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1, (Griffin,A. M., and Griffin, H. G., eds.), 1994, Humana Press, New Jersey; vonHeinje, G., SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, AcademicPress; SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.),1991, M. Stockton Press, New York; Carillo et al., 1988, SIAM J. AppliedMath., 48:1073; and Durbin et al., 1998, BIOLOGICAL SEQUENCE ANALYSIS,Cambridge University Press.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity aredescribed in publicly available computer programs. Preferred computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package, including GAP (Devereux etal., 1984, Nucl. Acid. Res., 12:387; Genetics Computer Group, Universityof Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul etal., 1990, J. Mol. Biol., 215:403-410). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., 1990, supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid or polynucleotidesequences may result in matching of only a short region of the twosequences, and this small aligned region may have very high sequenceidentity even though there is no significant relationship between thetwo full-length sequences. Accordingly, in certain embodiments, theselected alignment method (GAP program) will result in an alignment thatspans at least 50 contiguous amino acids of the target polypeptide. Insome embodiments, the alignment can comprise at least 60, 70, 80, 90,100, 110, or 120 amino acids of the target polypeptide. Ifpolynucleotides are aligned using GAP, the alignment can span at leastabout 100, 150, or 200 nucleotides, which can be contiguous.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is calculated as three-times the average diagonal; wherethe “average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually one-tenth of the gap openingpenalty), as well as a comparison matrix such as PAM250 or BLOSUM 62 areused in conjunction with the algorithm. In certain embodiments, astandard comparison matrix (see Dayhoff et al., 1978, Atlas of ProteinSequence and Structure, 5:345-352 for the PAM 250 comparison matrix;Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA, 89:10915-10919 forthe BLOSUM 62 comparison matrix) is also used by the algorithm.

In certain embodiments, the parameters for a polypeptide sequencecomparison include the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol, 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program may be useful with the above parameters. For nucleotidesequences, parameters can include a gap penalty of 50 and a gap lengthpenalty of 3, that is a penalty of 3 for each symbol in each gap. Incertain embodiments, the aforementioned parameters are the defaultparameters for polypeptide comparisons (along with no penalty for endgaps) using the GAP algorithm.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See IMMUNOLOGY—A SYNTHESIS, 2ndEdition, (E. S. Golub and D. R. Gren, Eds.), Sinauer Associates:Sunderland, Mass., 1991, incorporated herein by reference for anypurpose. Stereoisomers (e.g., D-amino acids) of the twenty conventionalamino acids; unnatural amino acids such as α-, α-disubstituted aminoacids, N-alkyl amino acids, lactic acid, and other unconventional aminoacids may also be suitable components for polypeptides of the invention.Examples of unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxyl-terminal direction, in accordancewith standard usage and convention.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: H is, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the human antibody that arehomologous with non-human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in certain embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In certain embodiments,those that are within ±1 are included, and in certain embodiments, thosewithin ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, asdisclosed herein. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those that are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions Exemplary Original ResiduesSubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn GluAsp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met,Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Val, Met, Ille Ala, PheLys Arg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile LeuPhe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr ThrScr Scr Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Set Phe Val Ile, Met, Leu,Phe, Ala, Leu Norleucine

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In other embodiments,the skilled artisan can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In further embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, the skilledartisan can predict the importance of amino acid residues in a proteinthat correspond to amino acid residues important for activity orstructure in similar proteins. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. In certain embodiments, one skilledin the art may choose to not make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays known to those skilled in theart. Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change can be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See, for example, Moult, 1996, Curr. Op. inBiotech. 7:422-427; Chou et al., 1974, Biochemistry 13:222-245; Chou etal., 1974, Biochemistry 113:211-222; Chou et al., 1978, Adv. Enzymol.Relat. Areas Mol. Biol. 47:45-148; Chou et al., 1979, Ann. Rev. Biochem.47:251-276; and Chou et al., 1979, Biophys. J. 26:367-384. Moreover,computer programs are currently available to assist with predictingsecondary structure. One method of predicting secondary structure isbased upon homology modeling. For example, two polypeptides or proteinsthat have a sequence identity of greater than 30%, or similarity greaterthan 40% often have similar structural topologies. The recent growth ofthe protein structural database (PDB) has provided enhancedpredictability of secondary structure, including the potential number offolds within a polypeptide's or protein's structure. See Holm et al.,1999, Nucl. Acid. Res. 27:244-247. It has been suggested (Brenner etal., 1997, Curr. Op. Struct. Biol. 7:369-376) that there are a limitednumber of folds in a given polypeptide or protein and that once acritical number of structures have been resolved, structural predictionwill become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskovet al., 1987, Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionarylinkage” (See Holm, 1999, supra; and Brenner, 1997, supra).

According to certain embodiments, amino acid substitutions are thosethat: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (5) confer ormodify other physicochemical or functional properties on suchpolypeptides. According to certain embodiments, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) may be made in the naturally occurring sequence (incertain embodiments, in the portion of the polypeptide outside thedomain(s) forming intermolecular contacts). In preferred embodiments, aconservative amino acid substitution typically does not substantiallychange the structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in PROTEINS,STRUCTURES AND MOLECULAR PRINCIPLES, (Creighton, Ed.), 1984, W. H.Freeman and Company, New York; INTRODUCTION TO PROTEIN STRUCTURE (C.Branden and J. Tooze, eds.), 1991, Garland Publishing, New York, N.Y.;and Thornton et al., 1991, Nature 354:105, each of which areincorporated herein by reference.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. See Fauchere, 1986, Adv. Drug Res.15:29; Veber & Freidinger, 1985, TINS p. 392; and Evans et al., 1987, J.Med. Chem. 30:1229, which are incorporated herein by reference for anypurpose. Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce a similartherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from: —CH₂—NH—, CH₂—S—, CH₂—CH₂—, CH═CH-(cis andtrans), —COCH₂—, CH(OH)CH₂—, and —CH₂SO—, by methods well known in theart. Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used in certain embodiments to generate more stablepeptides. In addition, constrained peptides comprising a consensussequence or a substantially identical consensus sequence variation maybe generated by methods known in the art (Rizo & Gierasch, 1992, Ann.Rev. Biochem. 61:387, incorporated herein by reference for any purpose);for example, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide.

As used herein, the terms “antibody” or “antibody peptide(s)” refer to amonomeric or multimeric protein comprising one or more polypeptidechains. An antibody can bind specifically to an antigen and may be ableto inhibit or modulate the biological activity of the antigen.“Antibodies” include naturally occurring antibodies, which are describedbelow. In certain embodiments, antibodies are produced by recombinantDNA techniques. In additional embodiments, antibodies are produced byenzymatic or chemical cleavage of naturally occurring antibodies.Antibodies include, but are not limited to, F(ab), F(ab′), F(ab′)₂, Fv,and single chain Fv fragments, as well as single-chain, chimeric,humanized, fully human, polyclonal, and monoclonal antibodies. At aminimum, an antibody, as meant herein, comprises a polypeptide that canbind specifically to an antigen comprising all or part of a light orheavy chain variable region.

A variable region comprises at least three heavy or light chaincomplementarity determining regions (CDRs, also known as hypervariableregions, designated CDR1, CDR2, and CDR3 by Kabat et al., 1991,Sequences of Proteins of Immunological Interest, Public Health ServiceN.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol.196: 901-17; Chothia et al., 1989, Nature 342: 877-83) embedded within aframework region (designated framework regions 1-4, FR1, FR2, FR3, andFR4, by Kabat et al., supra; see also Chothia and Lesk, supra). The CDRsand the framework segments are interspersed as follow, starting at theamino terminus of the variable region: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The primary sequences of the framework regions of antibody variableregions have a handful of residues that are universally conserved acrossphyla. However, many residues are highly conserved across phyla and/orwithin species and/or phyla, and many positions within antibodies areusually occupied by one of a known group of amino acids. See Kabat etal., supra. Alternatively a sequence can be recognized as an antibody byits predicted tertiary structure. The tertiary structure of the variableregions, which comprises 9β strands forming a structure known as a Greekkey β barrel, is extremely well conserved, and the positions of the CDRswithin this structure are also highly conserved. See e.g., Bork et al.,1994, J. Mol. Biol. 242: 309-20; Hunkapiller and Hood, 1989, Adv.Immunol. 44: 1-63; Williams and Barclay, 1988, Ann. Rev. Immunol. 6:381-405; Chothia and Lesk, supra; Kabat et al., supra.

Tertiary structure can be predicted by various computer programs, suchas, for example, GENEFOLD® (Tripos, Inc., St. Louis, Mo.; Godzik andSkolnik, 1992, Proc. Natl. Acad. Sci. USA 89: 12098-12102; Godzik etal., 1992, J. Mol. Biol. 227: 227-38; Godzik et al., 1993, ProteinEngng. 6: 801-10), a protein threading program that overlays a queryprotein sequence onto structural representatives of the Protein DataBank (PDB) (Berman et al., 2000, Nucleic Acids Res 28: 235-242;Jaroszewski et al., 1998, Prot Sci 7: 1431-1440). To use GENEFOLD® toclassify a new amino acid sequence, the sequence is entered into theprogram, which assigns a probability score that reflects how well itfolds onto previously known protein structures (“template” structures)that are present in the GENEFOLD® database. For scoring, GENEFOLD®relies on primary amino acid sequence similarity, burial patterns ofresidues, local interactions and secondary structure comparisons. TheGENEFOLD® program folds (or threads) the amino acid sequence onto all ofthe template structures in a preexisting database of protein folds,which includes the solved structures for a number of antibodies. Theoutput of GENEFOLD® is three lists of proteins from within the database,the tertiary structures of which are the most likely to be assumed bythe input amino acid sequence. The three lists contain three differentscores calculated based on (i) sequence only, (ii) sequence plus localconformation preferences plus burial terms, and (iii) sequence pluslocal conformation preferences plus burial terms plus secondarystructure. In each instance, the program determines the optimalalignment, calculates the probability (P-value) that this degree ofalignment occurred by chance, and reports the inverse of the P-value asthe score with 999.9 (9.999×10²) being the highest possible score. Thus,the highest score indicates the lowest probability that the alignmentoccurred by chance. These scores therefore reflect the degree to whichthe new protein matches the various reference structures and are usefulfor assigning a new protein to membership in a known family of proteins.For example, a sequence having the structure of an antibody variableregion would be expected to be aligned with at least one known antibodyvariable region with a reasonably high P-value, such as at least about200, 300, 400, 500, 600, 700, 800, or higher.

The term “heavy chain” includes any immunoglobulin polypeptide havingsufficient variable region sequence to confer binding specificity forIFN-γ. The term “light chain” includes any immunoglobulin polypeptidehaving sufficient variable region sequence to confer binding specificityfor IFN-γ. Such a heavy or light chain may, but need not, bind to IFN-γin the absence of a light chain, if it is a heavy chain, or a heavychain, if it is a light chain. A full-length heavy chain includes avariable region domain, V_(H), and three constant region domains,C_(H1), C_(H2), and C_(H3). The V_(H) domain is at the amino-terminus ofthe polypeptide, and the C_(H3) domain is at the carboxyl-terminus. Theterm “heavy chain”, as used herein, encompasses a full-length heavychain and fragments thereof. A full-length light chain includes avariable region domain, V_(L), and a constant region domain, C_(L). Likethe heavy chain, the variable region domain of the light chain is at theamino-terminus of the polypeptide. The term “light chain”, as usedherein, encompasses a full-length light chain and fragments thereof. AnF(ab) fragment is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of an F(ab)molecule cannot form a disulfide bond with another heavy chain molecule.An F(ab′) fragment contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form an F(ab′)₂ molecule. The Fv region comprisesthe variable regions from both the heavy and light chains, but lacks theconstant regions. Single-chain antibodies are Fv molecules in which theheavy and light chain variable regions have been connected by a flexiblelinker to form a single polypeptide chain, which forms anantigen-binding region. Single chain antibodies are discussed in detailin International Patent Application Publication No. WO 88/01649 and U.S.Pat. Nos. 4,946,778 and 5,260,203.

The invention also encompasses fully human, humanized, and chimericantibodies. As meant herein, fully human antibodies comprise amino acidsequences encoded only by polynucleotides that are ultimately of humanorigin or amino acid sequences that are identical to such sequences. Asmeant herein, antibodies encoded by human immunoglobulin-encoding DNAinserted into a mouse genome produced in a transgenic mouse are fullyhuman antibodies since they are encoded by DNA that is ultimately ofhuman origin. In this situation, human immunoglobulin-encoding DNA canbe rearranged (to encode an antibody) within the mouse, and somaticmutations may also occur. Antibodies encoded by originally human DNAthat has undergone such changes in a mouse are fully human antibodies asmeant herein. The use of such transgenic mice makes it possible toselect fully human antibodies against a human antigen. In nature, thisis not possible in most instances since a human immune response againsta self antigen does not normally occur. One of skill in the art willappreciate that fully human antibodies are advantageous for use astherapeutics, particularly to treat chronic diseases, since they areunlikely to precipitate an immune response against themselves. Incontrast, many non-human antibodies are known to precipitate an immuneresponse against themselves when used in humans, a situation that makeschronic use of such antibodies in humans inadvisable. Fully humanantibodies thus solve a long-standing problem faced in using antibodiesto treat chronic conditions, including human diseases. See e.g. Billiau,1988, Immunol. Today 9:37-40; Horneff et al., 1991, Clin. Immunol. &Immunopathol. 59:89-103; Tjandra et al., 1990, Immunol & Cell Biol.68:367-76. Therefore, fully human anti-IFN-γ antibodies are particularlywell suited for the treatment of chronic human IFN-γ mediated diseases,such as autoimmune diseases.

In a humanized antibody, the entire antibody, except the CDRs, isencoded by a polynucleotide of human origin or is identical to such anantibody except within its CDRs. The CDRs, which are encoded by nucleicacids originating in a non-human organism, are grafted into the β-sheetframework of a human antibody variable region to create an antibody, thespecificity of which is determined by the engrafted CDRs. The creationof such antibodies is described in, e.g., WO 92/11018, Jones et al.,1986, Nature 321:522-25, Verhoeyen et al., 1988, Science 239:1534-36.This work underlines the pivotal importance of the CDRs in forming anantigen binding site. A chimeric antibody comprises a human constantregion (which is encoded by a polynucleotide of human origin or isidentical to such an human constant region) and a non-human variableregion. The creation of such antibodies is described in, e.g., U.S. Pat.No. 5,681,722.

A bivalent antibody other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is understood to comprise bindingsites having identical antigenic specificity.

In assessing antibody binding and specificity according to theinvention, an antibody binds specifically and/or substantially inhibitsadhesion of a IFN-γ to its receptor when an excess of antibody reducesthe quantity of receptor bound to IFN-γ, or vice versa, by at leastabout 20%, 40%, 60%, 80%, 85%, or more (as measured in an in vitrocompetitive binding assay). A specifically-binding antibody can beexpected to have an equilibrium dissociation constant for binding toIFN-γ of less than or equal to than 10⁻⁸ molar, optionally less than orequal to 10⁻⁹ or 10⁻¹⁰ molar.

For therapeutic use, an important characteristic of an anti-IFN-γantibody is whether it can inhibit or modulate the biological activityof IFN-γ. IFN-γ has many distinct biological effects, which can bemeasured in many different assays in different cell types. The abilityof an anti-IFN-γ antibody to inhibit or modulate the biological activityof IFN-γ can be measured using the A549 assay described in Example 6below or using a similar assay in which the ability of an antibody toreverse the inhibition of cell proliferation observed in the presence ofIFN-γ is measured. For the assay to produce meaningful results, theproliferation of the cells used in the assay must be inhibited by theIFN-γ used in the assay. Human IFN-γ can inhibit proliferation of somecell types, including A549 cells (Examples 6 and 7). Murine IFN-γ caninhibit the proliferation of RAW 264.7 cells (Example 7), but not A549cells. Especially when testing the ability of an antibody to inhibit ormodulate the biological activity of a non-human IFN-γ cell types otherthan A549 cells can be used since non-human IFN-γ may or may not be ableto inhibit proliferation of A549 cells. Not every antibody thatspecifically binds to an antigen can block antigen binding to its normalreceptor and thus inhibit or modulate the biological effects of theantigen upon binding to its receptor. As is known in the art, such aneffect can depend on what portion of the antigen the antibody binds toand on the both the absolute and the relative concentrations of theantigen and the antibody, in this case, IFN-γ and the anti-IFN-γantibody. To be considered capable of inhibiting or modulating thebiological activity of IFN-γ as meant herein, an antibody must be ableto reverse the inhibition of cell proliferation observed in the presenceof IFN-γ, as measured by fluorescence in the A549 assay (Example 6) or asimilar assay, by at least about 20%, 40%, 60%, 80%, 85%, 100%, or morewhen the IFN-γ concentration is within a range, for example, at aboutEC₈₀ or EC₉₀, where the effects of an agent that inhibits its biologicalactivity can be readily apparent. An EC₈₀, as meant herein, is theamount of IFN-γ required for 80% of the maximal effect of IFN-γ to beobserved. If the IFN-γ concentration is well above EC₉₀, effects of aninhibiting agent may be less apparent due to the excess of IFN-γ. Theconcentration of an antibody required to inhibit or modulate thebiological activity of IFN-γ can vary widely and may depend upon howtightly the antibody binds to IFN-γ. For example, one molecule or lessof an antibody per molecule of IFN-γ may be sufficient to inhibit ormodulate biological activity in the A549 assay. In some embodiments, aratio of IFN-γ antibody of about 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10,1:20, 1:40, 1:60, 1:100, or 1:50,000 may be required to inhibit ormodulate the biological activity of IFN-γ when the IFN-γ concentrationis from about EC₅₀ to about EC₉₀. Ratios of IFN-γ:antibody between thesevalues are also possible.

In additional embodiments, antibody variants can include antibodiescomprising a modified Fc fragment or a modified heavy chain constantregion. An Fc fragment, which stands for “fragment that crystallizes,”or a heavy chain constant region can be modified by mutation to conferon an antibody altered characteristics. See, for example, Burton andWoof, 1992, Advances in Immunology 51: 1-84; Ravetch and Bolland, 2001,Annu. Rev. Immunol. 19: 275-90; Shields et al., 2001, Journal of Biol.Chem. 276: 6591-6604; Telleman and Junghans, 2000, Immunology 100:245-251; Medesan et al., 1998, Eur. J. Immunol. 28: 2092-2100; all ofwhich are incorporated herein by reference). Such mutations can includesubstitutions, additions, deletions, or any combination thereof, and aretypically produced by site-directed mutagenesis using one or moremutagenic oligonucleotide(s) according to methods described herein, aswell as according to methods known in the art (see, for example,Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed., 2001,Cold Spring Harbor, N.Y. and Berger and Kimmel, METHODS IN ENZYMOLOGY,Volume 152, Guide to Molecular Cloning Techniques, 1987, Academic Press,Inc., San Diego, Calif., which are incorporated herein by reference).

In certain embodiments, antibody variants include glycosylation variantswherein the number and/or type of glycosylation site has been alteredcompared to the amino acid sequences of the parent polypeptide. Incertain embodiments, protein variants comprise a greater or a lessernumber of N-linked glycosylation sites than the native protein. AnN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Asn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. Additional preferred antibodyvariants include cysteine variants wherein one or more cysteine residuesare deleted from or substituted for another amino acid (e.g., serine)compared to the parent amino acid sequence. Cysteine variants may beuseful when antibodies must be refolded into a biologically activeconformation such as after the isolation of insoluble inclusion bodies.Cysteine variants generally have fewer cysteine residues than the nativeprotein, and typically have an even number to minimize interactionsresulting from unpaired cysteines.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid, or attachment to a polypeptide or nucleic acid of a fluorescentmarker, a chemiluminescent marker or an enzyme having a detectableactivity, or attachment to a polypeptide of biotin moieties that can bedetected by labeled avidin (e.g., streptavidin preferably comprising adetectable marker such as a fluorescent marker, a chemiluminescentmarker or an enzymatic activity that can be detected, inter alia, byoptical or colorimetric methods). In certain embodiments, the label canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used advantageously in themethods disclosed herein. Examples of labels for polypeptides include,but are not limited to, the following: radioisotopes or radionuclides(e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ^(99m)Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescentlabels (e.g., fluorescein isothiocyanate or FITC, rhodamine, orlanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentlabels, hapten labels such as biotinyl groups, and predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, or epitope tags). In certain embodiments, labels areattached by spacer arms (such as (CH₂)_(n), where n<about 20) of variouslengths to reduce potential steric hindrance.

The term “biological sample”, as used herein, includes, but is notlimited to, any quantity of a substance from a living thing or formerlyliving thing. Such living things include, but are not limited to,humans, mice, monkeys, rats, rabbits, and other animals. Such substancesinclude, but are not limited to, blood, serum, urine, cells, organs,tissues, bone, bone marrow, lymph nodes, and skin.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient.

The term “IFN-γ mediated disease” includes, but is not limited to,inflammatory, infectious, and autoimmune diseases. An “autoimmunedisease” as used herein refers to disease states and conditions whereina patient's immune response is directed toward the patient's ownconstituents. For example, IFN-γ mediated diseases include, but are notlimited to, Acquired Immune Deficiency Syndrome (AIDS), rheumatoidarthritis including juvenile rheumatoid arthritis, inflammatory boweldiseases including ulcerative colitis and Crohn's disease, multiplesclerosis, Addison's disease, diabetes (type I), epididymitis,glomerulonephritis, Graves' disease, Guillain-Barre syndrome,Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus(SLE), lupus nephritis, myasthenia gravis, pemphigus, psoriasis,psoriatic arthritis, atherosclerosis, erythropoietin resistance, graftversus host disease, transplant rejection, autoimmune hepatitis-inducedhepatic injury, biliary cirrhosis, alcohol-induced liver injuryincluding alcoholic cirrhosis, rheumatic fever, sarcoidosis,scleroderma, Sjogren's syndrome, spondyloarthropathies includingankylosing spondylitis, thyroiditis, and vasculitis. The term “IFN-gammamediated disease” also encompasses any medical condition associated withincreased levels of IFN-γ or increased sensitivity to IFN-γ.

Treatment of an IFN-γ mediated disease, including an autoimmune disease,encompasses alleviation of at least one symptom of the disorder, areduction in the severity of the disease, or the delay or prevention ofprogression to a more serious disease that occurs with some frequencyfollowing the treated condition. Treatment need not mean that thedisease is totally cured. A useful therapeutic agent needs only toreduce the severity of a disease, reduce the severity of a symptom orsymptoms associated with the disease or its treatment, or provideimprovement to a patient's quality of life, or delay the onset of a moreserious disease that can occur with some frequency following the treatedcondition. For example, if the disease is a rheumatoid arthritis, atherapeutic agent may decrease swelling of joints, reduce the number ofjoints affected, or delay or inhibit bone loss. An SLE patient can havesymptoms such as skin lesions, fever, weakness, arthritis,lymphadenopathy, pleurisy, pericarditis, and/or anemia, among others.Such symptoms can be assessed by any of a number of conventionaltechniques including, for example, visual observation, photography,measurement of temperature, grip strength, or joint size, and/ormicroscopic examination of blood to determine the concentration of redblood cells. The invention encompasses a method of treatment comprisingadministering to a patient afflicted with a IFN-γ mediated disease anIFN-γ antibody of the invention in an amount and for a time sufficientto induce a sustained improvement over baseline of an indicator thatreflects the severity of a particular disorder or the severity ofsymptoms caused by the disorder or to delay or prevent the onset of amore serious disease that follows the treated condition in some or allcases. The invention does not exclude possible treatment with othertherapeutic agents before, after, and/or during treatment with the IFN-γantibody.

As used herein, “substantially pure” or “substantially purified” means acompound or species that is the predominant species present (i.e., on amolar basis it is more abundant than any other individual species in thecomposition). In certain embodiments, a substantially purified fractionis a composition wherein the species comprises at least about 50 percent(on a molar basis) of all macromolecular species present. In certainembodiments, a substantially pure composition will comprise more thanabout 80%, 85%, 90%, 95%, or 99% of all macromolar species present inthe composition. In certain embodiments, the species is purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species.

The term “patient” includes human and animal subjects.

Unless otherwise required by context, singular terms shall includepluralities.

Because IFN-γ is a cytokine with multiple functions, includingprotecting the body from viral infection and regulating several aspectsof the immune response, increased IFN-γ activity can contribute toseveral pathological conditions. According to certain embodiments of theinvention, antibodies directed to IFN-γ may be used to treat IFN-γmediated diseases, including but not limited to, those mentioned above.

In one aspect of the invention are provided fully human monoclonalantibodies raised against and having biological and immunologicalspecificity for specific binding to human IFN-γ. Variable regions (SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:30, and SEQ ID NO:31) included in such antibodies,complete heavy and light chains of such antibodies (SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22), andantibodies comprising specific CDRs (heavy and light chain CDR1, CDR2,and/or CDR3; SEQ ID NO:34 through SEQ ID NO:44) are encompassed by theinvention. Particular embodiments of this aspect of the invention aresequences corresponding to CDR's, specifically from CDR1 through CDR3,of the heavy and light chains provided by the invention. Further, theinvention encompasses antibodies comprising a CDR3 sequence disclosedherein (SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:43, and/or SEQ ID NO:44)that may also contain sequences at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%. 96%, 97%, 98%, or about 99% identical to any of the variableregion sequences or complete heavy or light chain sequences disclosedherein, wherein the antibody can inhibit or modulate the biologicalactivity of IFN-γ.

In another aspect the invention provides isolated nucleic acids orpolynucleotides encoding the antibodies of the invention. Antibodies ofthe invention can bind specifically to and/or inhibit or modulate thebiological activity of IFN-γ. Specifically encompassed by the inventionare polynucleotides comprising nucleotide sequences encoding the aminoacid sequences SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:30, SEQ ID NO:31, and/or sequences that are at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% identical to thesesequences, wherein the alignment between the sequences above and thetest sequence span at least about 50, 60, 70, 80, 90, or 100 aminoacids. The invention further provides polynucleotides comprising SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:48, that encodeantibodies that can bind specifically to and/or inhibit or modulate thebiological activity of IFN-γ. Further, the invention encompassespolynucleotides that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or about 99% identical to SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:30, SEQID NO:31, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:32, or SEQ ID NO:33, wherein an antibodyencoded in part by each of these polynucleotides can inhibit or modulatethe biological activity of and/or bind specifically to IFN-γ and whereinthe alignment between the nucleotide sequences named immediately aboveand the test sequence spans at least about 100, 150, or 200 nucleotides.

Table 2 provides a brief description of the sequences as they relate totheir sequence identification numbers.

TABLE 2 BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS SequenceIdentification Number Brief Description SEQ ID NO: 1 Nucleotide sequenceencoding the heavy chain constant region of the 1118, 1118*, 1119, 1121,or 1121* antibody SEQ ID NO: 2 Amino acid sequence of the heavy chainconstant region of the 1118, 1118*, 1119, 1121, or 1121* antibody SEQ IDNO: 3 Nucleotide sequence encoding the light chain constant region ofthe 1118, 1118*, 1119, 1121, or 1121* antibody SEQ ID NO: 4 Amino acidsequence of the light chain constant region of the 1118, 1118*, 1119,1121, or 1121* antibody SEQ ID NO: 5 Nucleotide sequence encoding theheavy chain variable region of the 1119 antibody SEQ ID NO: 6 Amino acidsequence of the heavy chain variable region of the 1119 antibody SEQ IDNO: 7 Nucleotide sequence encoding the light chain variable region ofthe 1119 antibody SEQ ID NO: 8 Amino acid sequence of the light chainvariable region of the 1119 antibody SEQ ID NO: 9 Nucleotide sequenceencoding the heavy chain variable region of the 1118 antibody SEQ ID NO:10 Amino acid sequence of the heavy chain variable region of the 1118antibody SEQ ID NO: 11 Nucleotide sequence encoding the light chainvariable region of the 1118 or 1118* antibody SEQ ID NO: 12 Amino acidsequence of the light chain variable region of the 1118 or 1118*antibody SEQ ID NO: 13 Nucleotide sequence encoding the heavy chainvariable region of the 1121 or 1121* antibody SEQ ID NO: 14 Amino acidsequence of the heavy chain variable region of the 1121 or 1121*antibody SEQ ID NO: 15 Nucleotide sequence encoding the light chainvariable region of the 1121 antibody SEQ ID NO: 16 Amino acid sequenceof the light chain variable region of the 1121 antibody SEQ ID NO: 17Amino acid sequence of the entire heavy chain of the 1119 antibody SEQID NO: 18 Amino acid sequence of the entire light chain of the 1119antibody SEQ ID NO: 19 Amino acid sequence of the entire heavy chain ofthe 1118 antibody SEQ ID NO: 20 Amino acid sequence of the entire lightchain of the 1118 or 1118* antibody SEQ ID NO: 21 Amino acid sequence ofthe entire heavy chain of the 1121 or 1121* antibody SEQ ID NO: 22 Aminoacid sequence of the entire light chain of the 1121 antibody SEQ ID NO:23 Nucleotide sequence of a PCR primer SEQ ID NO: 24 Nucleotide sequenceof a PCR primer SEQ ID NO: 25 Nucleotide sequence of a PCR primer SEQ IDNO: 26 Nucleotide sequence of a PCR primer SEQ ID NO: 27 Nucleotidesequence of a PCR primer SEQ ID NO: 28 Nucleotide sequence of a PCRprimer SEQ ID NO: 29 Nucleotide sequence of a PCR primer SEQ ID NO: 30Amino acid sequence of the heavy chain variable region of 1118* antibodySEQ ID NO: 31 Amino acid sequence of the light chain variable region of1121* antibody SEQ ID NO: 32 Amino acid sequence of the entire heavychain of the 1118* antibody SEQ ID NO: 33 Amino acid sequence of theentire light chain of the 1121* antibody SEQ ID NO: 34 Amino acidsequence of the heavy chain CDR1 of the 1119, 1118, 1118*, 1121, or1121* antibody SEQ ID NO: 35 Amino acid sequence of the heavy chain CDR2of the 1119, 1118, 1118*, 1121, or 1121* antibody SEQ ID NO: 36 Aminoacid sequence of the heavy chain CDR3 of the 1119 antibody SEQ ID NO: 37Amino acid sequence of the heavy chain CDR3 of the 1118, 1118*, 1121, or1121* antibody SEQ ID NO: 38 Amino acid sequence of the light chain CDR1of the 1119 or 1121 antibody SEQ ID NO: 39 Amino acid sequence of thelight chain CDR1 of the 1118 or 1118* antibody SEQ ID NO: 40 Amino acidsequence of the light chain CDR1 of the 1121* antibody SEQ ID NO: 41Amino acid sequence of the light chain CDR2 of the 1119, 1118, 1118*, or1121 antibody SEQ ID NO: 42 Amino acid sequence of the light chain CDR2of the 1121* antibody SEQ ID NO: 43 Amino acid sequence of the lightchain CDR3 of the 1119, 1118, 1118*, or 1121 antibody SEQ ID NO: 44Amino acid sequence of the light chain CDR3 of the 1121* antibody SEQ IDNO: 45 Nucleotide sequence encoding the heavy chain CDR3 of the 1119antibody SEQ ID NO: 46 Nucleotide sequence encoding the heavy chain CDR3of the 1118, 1118*, 1121, or 1121* antibody SEQ ID NO: 47 Nucleotidesequence encoding the light chain CDR3 of the 1118, 1118*, 1119, or 1121antibody SEQ ID NO: 48 Amino acid sequence immediately preceding a heavychain CDR1 SEQ ID NO: 49 Amino acid sequence that may immediatelyprecede a heavy chain CDR2 SEQ ID NO: 50 Amino acid sequence that almostalways follows a heavy chain CDR3 SEQ ID NO: 51 Amino acid sequence thatusually follows a light chain CDR3 SEQ ID NO: 52 Amino acid sequence ofa signal sequence SEQ ID NO: 53 Amino acid sequence of a signal sequenceSEQ ID NO: 54 Amino acid sequence of a signal sequence SEQ ID NO: 55Amino acid sequence of a signal sequence SEQ ID NO: 56 Nucleotidesequence of the heavy chain variable region of 1118* antibody SEQ ID NO:57 Nucleotide sequence of the light chain variable region of 1121*antibody

In yet another aspect the invention provides hybridoma cells and celllines that express the immunoglobulin molecules and antibodies of theinvention, optionally monoclonal antibodies. In a further aspect, ahybridoma cell or a cell from a cell line that expresses and/or secretesan immunoglobulin molecule or antibody of the invention can be implantedin a patient, whereby an antibody of the invention or immunologicallyfunctional immunoglobulin fragment thereof is expressed and/or secretedin the patient, thereby inhibiting or modulating IFN-γ activity.

The invention also provides biologically and immunologically purifiedpreparations of antibodies, preferably monoclonal antibodies raisedagainst and having biological and immunological specificity for bindingspecifically to human IFN-γ.

The ability to clone and reconstruct megabase-sized human loci in yeastartificial chromosomes (YACs) and to introduce them into the mousegermline permits development of an advantageous approach to elucidatingthe functional components of very large or crudely mapped loci as wellas generating useful models of human disease. Furthermore, theutilization of such technology for substitution of mouse loci with theirhuman equivalents produces unique insights into the expression andregulation of human gene products during development, theircommunication with other systems, and their involvement in diseaseinduction and progression.

An important practical application of such a strategy is the alterationof the mouse humoral immune system by the introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated. International Application No. WO 93/12227. This systemoffers the opportunity to study mechanisms underlying programmedexpression and assembly of antibodies as well as their role in B-celldevelopment. Furthermore, such a strategy provides a source forproduction of fully human monoclonal antibodies (MAbs). Fully human MAbsare expected to minimize the immunogenic and allergic responsesintrinsic to mouse or mouse-derived MAbs, and to thereby increase theefficacy and safety of the administered antibodies. Fully humanantibodies can be used in the treatment of chronic and recurring humandiseases, such as osteoarthritis, rheumatoid arthritis, and otherinflammatory conditions, the treatment thereof requiring repeatedantibody administration. Thus, one particular advantage of theanti-IFN-γ antibodies of the invention is that the antibodies are fullyhuman and can be administered to patients in a non-acute manner whileminimizing adverse reactions commonly associated with mouse anti-humanantibodies or other previously described non-fully human antibodies ornon-human antibodies from non-human species.

Using methods set forth herein, one skilled in the art can engineermouse strains deficient in mouse antibody production with largefragments of the human Ig loci so that such mice produce humanantibodies in the absence of mouse antibodies. Large human Ig fragmentsmay preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse cellular machinery for antibody diversification and selection andthe lack of immunological tolerance to human proteins, the reproducedhuman antibody repertoire in these mouse strains yields high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human MAbs with thedesired specificity may be produced and selected.

In certain embodiments, the skilled artisan can use constant regionsfrom species other than human along with the human variable region(s) insuch mice to produce chimeric antibodies.

Naturally Occurring Antibody Structure

Most naturally occurring antibody structural units typically comprise atetramer. Each such tetramer typically is composed of two identicalpairs of polypeptide chains, each pair having one full-length “light”chain (typically having a molecular weight of about 25 kDa) and onefull-length “heavy” chain (typically having a molecular weight of about50-70 kDa). The amino-terminal portion of each light and heavy chaintypically includes a variable region of about 100 to 110 or more aminoacids that typically is responsible for antigen recognition. Thecarboxy-terminal portion of each chain typically defines a constantregion responsible for effector function. Human light chains aretypically classified as kappa and lambda light chains. Heavy chains aretypically classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgGhas several subclasses, including, but not limited to, IgG1, IgG2, IgG3,and IgG4. IgM has subclasses including, but not limited to, IgM1 andIgM2. IgA is similarly subdivided into subclasses including, but notlimited to, IgA1 and IgA2. Within full-length light and heavy chains,typically, the variable and constant regions are joined by a “J” regionof about 12 or more amino acids, with the heavy chain also including a“D” region of about 10 more amino acids. See, e.g., FUNDAMENTALIMMUNOLOGY, Ch. 7, 2^(nd) ed., (Paul, W., ed.), 1989, Raven Press, N.Y.(incorporated by reference in its entirety for all purposes). Thevariable regions of each light/heavy chain pair typically form theantigen-binding site.

Some naturally-occurring antibodies, which have been found in camels andllamas, are dimers consisting of two heavy chains and include no lightchains. Muldermans et al., 2001, J. Biotechnol. 74:277-302; Desmyter etal., 2001, J. Biol. Chem. 276:26285-90. The invention encompassesdimeric antibodies consisting of two heavy chains that can bind toand/or inhibit the biological activity of IFN-γ. A crystallographicstudy of a camel antibody has revealed that the heavy chain CDR3, whichis 19 amino acids long, forms a surface that interacts with the antigenand covers the two other hypervariable regions. Desmyter et al., supra.Thus, CDR3 is important for antigen binding in dimeric camel antibodies,as well as in the more typical tetrameric antibodies.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are typicallyembedded within the framework regions, which may enable binding to aspecific epitope. From N-terminal to C-terminal, both light and heavychain variable regions typically comprise the domains FR1, CDR1, FR2,CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain istypically in accordance with the definitions of Kabat et al., asexplained in more detail below. Kabat et al., Sequences of Proteins ofImmunological Interest (1991, National Institutes of Health, Bethesda,Md.); see also Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothiaet al., 1989, Nature 342:878-883. CDRs constitute the major surfacecontact points for antigen binding. See e.g. Chothia and Lesk, supra.Further, CDR3 of the light chain and, especially, CDR3 of the heavychain may constitute the most important determinants in antigen bindingwithin the light and heavy chain variable regions. See e.g. Chothia andLesk, supra; Desiderio et al. (2001), J. Mol. Biol. 310: 603-15; Xu andDavis (2000), Immunity 13(1): 37-45; Desmyter et al. (2001), J. Biol.Chem. 276(28): 26285-90; and Muyldermans (2001), J. Biotechnol. 74(4):277-302. In some antibodies, the heavy chain CDR3 appears to constitutethe major area of contact between the antigen and the antibody. Desmyteret al, supra. In vitro selection schemes in which CDR3 alone is variedcan be used to vary the binding properties of an antibody. Muyldermans,supra; Desiderio, supra.

CDRs can be located in a heavy chain variable region sequence in thefollowing way. CDR₁ starts at approximately residue 31 of the matureantibody and is usually about 5-7 amino acids long, and it is almostalways preceded by a Cys-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx (SEQ ID NO: 48)(where “Xxx” is any amino acid). The residue following the heavy chainCDR1 is almost always a tryptophan, often a Typ-Val, a Trp-Ile, or aTrp-Ala. Fourteen amino acids are almost always between the last residuein CDR1 and the first in CDR2, and CDR2 typically contains 16 to 19amino acids. CDR2 may be immediately preceded by Leu-Glu-Trp-Ile-Gly(SEQ ID NO: 49) and may be immediately followed byLys/Arg-Leu/IleNal/Phe/Thr/Ala-Thr/Ser/Ile/Ala. Other amino acids mayprecede or follow CDR2. Thirty-two amino acids are almost always betweenthe last residue in CDR2 and the first in CDR3, and CDR3 can be fromabout 3 to 25 residues long. A Cys-Xxx-Xxx almost always immediatelyprecedes CDR3, and a Trp-Gly-Xxx-Gly (SEQ ID NO: 50) almost alwaysfollows CDR3.

Light chain CDRs can be located in a light chain sequence in thefollowing way. CDR1 starts at approximately residue 24 of the matureantibody and is usually about 10 to 17 residues long. It is almostalways preceded by a Cys. There are almost always 15 amino acids betweenthe last residue of CDR1 and the first residue of CDR2, and CDR2 isalmost always 7 residues long. CDR2 is typically preceded by Ile-Tyr,Val-Tyr, Ile-Lys, or Ile-Phe. There are almost always 32 residuesbetween the light chain CDR2 and CDR3, and CDR3 is usually about 7 to 10amino acids long. CDR3 is almost always preceded by Cys and usuallyfollowed by Phe-Gly-Xxx-Gly (SEQ ID NO: 51).

One of skill in the art will realize that the lengths of frameworkregions surrounding the CDRs can contain insertions or deletions thatmake their length differ from what is typical. As meant herein, thelength of heavy chain framework regions fall within the followingranges: FR1, 0 to 41 amino acids; FR2, 5 to 24 amino acids; FR3, 13 to42 amino acids; and FR4, 0 to 21 amino acids. Further, the inventioncontemplates that the lengths of light chain framework regions fallwithin the following ranges: FR1, 6 to 35 amino acids; FR2, 4 to 25amino acids; FR3, 2 to 42 amino acids; and FR4, 0 to 23 amino acids.

Naturally occurring antibodies typically include a signal sequence,which directs the antibody into the cellular pathway for proteinsecretion and which is not present in the mature antibody. Apolynucleotide encoding an antibody of the invention may encode anaturally occurring signal sequence or a heterologous signal sequence asdescribed below.

In Vitro Maturation of Antibodies

Antibodies can be matured in vitro to produce antibodies with alteredproperties, such as a higher affinity for an antigen or a lowerdissociation constant. Variation of only residues within the CDRs,particularly the CDR3s, can result in altered antibodies that bind tothe same antigen, but with greater affinity. See e.g. Schier et al.,1996, J. Mol. Biol. 263:551-67; Yang et al., 1995, J. Mol. Biol.254:392-403. The invention encompasses antibodies created by a varietyof in vitro selection schemes, such as affinity maturation and/or chainshuffling (Kang et al., 1991, Proc. Natl. Acad. Sci. 88:11120-23), orDNA shuffling (Stemmer, 1994, Nature 370:389-391), by which antibodiesmay be selected to have advantageous properties. In many schemes, aknown antibody is randomized at certain positions, often within theCDRs, in vitro and subjected to a selection process whereby antibodieswith desired properties, such as increased affinity for a certainantigen, can be isolated. See e.g. van den Beucken et al., 2001, J. Mol.Biol. 310:591-601; Desiderio et al., 2001, J. Mol. Biol. 310:603-15;Yang et al., 1995, J. Mol. Biol. 254:392-403; Schier et al., 1996, J.Mol. Biol. 263:551-67. Typically, such mutated antibodies may compriseseveral altered residues in one or more CDRs, depending on the design ofthe mutagenesis and selection steps. See e.g. van den Beucken et al.,supra.

Bispecific or Bifunctional Antibodies

A bispecific or bifunctional antibody typically is an artificial hybridantibody having two different heavy chain/light chain pairs and twodifferent binding sites. Bispecific antibodies may be produced by avariety of methods including, but not limited to, fusion of hybridomasor linking of F(ab′) fragments. See, e.g., Songsivilai & Lachmann, 1990,Clin. Exp. Immunol. 79: 315-321; Kostelny et al., 1992, J. Immunol.148:1547-1553.

Preparation of Antibodies

The invention provides antibodies that bind specifically to human IFN-γ.These antibodies can be produced by immunization with full-length IFN-γor fragments thereof. The antibodies of the invention can be polyclonalor monoclonal and/or may be recombinant antibodies. In certainembodiments, fully human antibodies of the invention are prepared, forexample, by immunization of transgenic animals capable of producinghuman antibodies (see, for example, International Patent Application,Publication WO 93/12227).

The CDRs of the light chain and heavy chain variable regions ofanti-IFN-γ antibodies of the invention can be grafted to frameworkregions (FRs) from the same, or another, species. In certainembodiments, the CDRs of the light chain and heavy chain variableregions of anti-IFN-γ antibody may be grafted to consensus human FRs tocreate a “humanized” antibody. Such humanized antibodies are encompassedby the instant invention. To create consensus human FRs, FRs fromseveral human heavy chain or light chain amino acid sequences arealigned to identify a consensus amino acid sequence. The FRs of theanti-IFN-γ antibody heavy chain or light chain can be replaced with theFRs from a different heavy chain or light chain. Rare amino acids in theFRs of the heavy and light chains of anti-IFN-γ antibody typically arenot replaced, while the rest of the FR amino acids can be replaced. Rareamino acids are specific amino acids that are in positions in which theyare not usually found in FRs. The grafted variable regions fromanti-IFN-γ antibodies of the invention can be used with a constantregion that is different from an original constant region of ananti-IFN-γ antibody. Alternatively, the grafted variable regions arepart of a single chain Fv antibody. CDR grafting is described, e.g., inU.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and5,530,101, which are hereby incorporated by reference for any purpose.

Antibodies of the invention can be prepared using transgenic mice thathave a substantial portion of the human antibody producing locusinserted in antibody-producing cells of the mice, and that are furtherengineered to be deficient in producing endogenous, murine, antibodies.Such mice are capable of producing human immunoglobulin molecules andantibodies and do not produce or produce substantially reduced amountsof murine immunoglobulin molecules and antibodies. Technologies utilizedfor achieving this result are disclosed in the patents, applications,and references disclosed in the specification herein. In certainembodiments, the skilled worker may employ methods as disclosed inInternational Patent Application Publication No. WO 98/24893, which ishereby incorporated by reference for any purpose. See also Mendez etal., 1997, Nature Genetics 15:146-156, which is hereby incorporated byreference for any purpose.

The monoclonal antibodies (mAbs) of the invention can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975, Nature 256:495). Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibodies can be employed, e.g., viral oroncogenic transformation of B-lymphocytes.

One possible animal system for preparing hybridomas is the mouse.Hybridoma production in the mouse is very well established, andimmunization protocols and techniques for isolation of immunizedsplenocytes for fusion are well known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

In some embodiments fully human monoclonal antibodies directed againstIFN-γ, optionally human IFN-γ, can be generated using transgenic micecarrying parts of the human immune system rather than the mouse system.These transgenic mice, referred to herein as “HuMab” mice, contain ahuman immunoglobulin gene minilocus that encodes unrearranged humanheavy (μ and γ) and .kappa. light chain immunoglobulin sequences,together with targeted mutations that inactivate the endogenous μ and.kappa. chain loci (Lonberg et al., 1994, Nature 368:856-859).Accordingly, the mice exhibit reduced expression of mouse IgM or .kappa.and in response to immunization, the introduced human heavy chain andlight chain transgenes undergo class switching and somatic mutation togenerate high affinity human IgG .kappa. monoclonal antibodies (Lonberget al., supra.; Lonberg and Huszar, 1995, Intern. Rev. Immunol.13:65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci. 764:536-546).The preparation of HuMab mice is described in detail in Taylor et al.,1992, Nucleic Acids Res. 20:6287-6295; Chen et al., 1993, InternationalImmunology 5:647-656; Tuaillon et al., 1994, J. Immunol. 152:2912-2920;Lonberg et al., 1994, Nature 368:856-859; Lonberg, 1994, Handbook ofExp. Pharmacology 113:49-101; Taylor et al., 1994, InternationalImmunology 6:579-591; Lonberg & Huszar, 1995, Intern. Rev. Immunol.13:65-93; Harding & Lonberg, 1995, Ann. N.Y. Acad. Sci. 764:536-546;Fishwild et al., 1996, Nature Biotechnology 14:845-851, the contents ofall of which are hereby incorporated by reference in their entirety. Seefurther U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay, as well as U.S. Pat. No. 5,545,807 to Surani etal.; International Patent Application Publication Nos. WO 93/1227,published Jun. 24, 1993; WO 92/22646, published Dec. 23, 1992; and WO92/03918, published Mar. 19, 1992, the disclosures of all of which arehereby incorporated by reference in their entirety. Alternatively, theHCo7 and HCo12 transgenic mice strains described in the Examples belowcan be used to generate human anti-IFN-γ antibodies.

In these embodiments, the antibodies of the invention bind specificallyto IFN-γ with an equilibrium dissociation constant (K_(D)) of less than10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, or 10⁻¹⁰ M. In certain embodiments of theinvention, the antibodies bind to IFN-γ with a K_(D) of betweenapproximately 10⁸ M and 10¹² M.

In preferred embodiments, the antibodies of the invention are of theIgG1, IgG2, or IgG4 isotype. The antibodies can be of the IgG1 isotype.In other embodiments, the antibodies of the invention are of the IgM,IgA, IgE, or IgD isotype. In preferred embodiments of the invention, theantibodies comprise a human kappa light chain and a human IgG1 heavychain. Expression of antibodies of the invention comprising an IgG1heavy chain constant region is described in the Examples below. Inparticular embodiments, the variable regions of the antibodies areligated to a constant region other than the constant region for the IgG1isotype. In certain embodiments, the antibodies of the invention havebeen cloned for expression in mammalian cells.

In certain embodiments, conservative modifications to the heavy chainsand light chains of anti-IFN-γ antibody (and corresponding modificationsto the encoding nucleotides) will produce anti-IFN-γ antibodies havingfunctional and chemical characteristics similar to those of anti-IFN-γantibody. In contrast, substantial modifications in the functionaland/or chemical characteristics of anti-IFN-γ antibody may beaccomplished by selecting substitutions in the amino acid sequence ofthe heavy and light chains that differ significantly in their effect onmaintaining (a) the structure of the molecular backbone in the area ofthe substitution, for example, as a β sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a normative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. In certain embodiments, amino acidsubstitutions can be used to identify important residues of anti-IFN-γantibody, or to increase or decrease the affinity of the anti-IFN-γantibodies described herein.

In alternative embodiments, antibodies of the invention can be expressedin cell lines other than hybridoma cell lines. In these embodiments,sequences encoding particular antibodies can be used for transformationof a suitable mammalian host cell. According to these embodiments,transformation can be achieved using any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455 (all of which are hereby incorporated herein byreference for any purpose). Generally, the transformation procedure usedmay depend upon the host to be transformed. Methods for introducingheterologous polynucleotides into mammalian cells are well known in theart and include, but are not limited to, dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Nucleic acid molecules (or polynucleotides) encoding the amino acidsequence of a heavy chain constant region, a heavy chain variableregion, a light chain constant region, or a light chain variable regionof an anti-IFN-γ antibody of the invention are encompassed by theinvention. Such polynucleotides can be inserted into an appropriateexpression vector using standard ligation techniques. In a preferredembodiment, a polynucleotide encoding the anti-IFN-γ antibody heavychain or light chain constant region is appended to the downstream endof a polynucleotide encoding the appropriate variable region and isligated into an expression vector. The vector is typically selected tobe functional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). For a review ofexpression vectors, see METH. ENZ. 185 (Goeddel, ed.), 1990, AcademicPress.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the anti-IFN-γantibody polypeptide coding sequence; the oligonucleotide sequenceencodes polyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the IFN-γ antibody from the host cell.Affinity purification can be accomplished, for example, by columnchromatography using antibodies against the tag as an affinity matrix.Optionally, the tag can subsequently be removed from the purifiedanti-IFN-γ antibody polypeptide by various means such as using certainpeptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (ie., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagene® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantibody that binds to IFN-γ polypeptide. As a result, increasedquantities of a polypeptide such as an anti-IFN-γ antibody aresynthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgamo sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addpro-sequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain apromoter that is recognized by the host organism and operably linked tothe molecule encoding the anti-IFN-γ antibody. Promoters areuntranscribed sequences located upstream (i.e., 5′) to the start codonof a structural gene (generally within about 100 to 1000 bp) thatcontrol transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,uniformly transcribe gene to which they are operably linked, that is,with little or no control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A suitable promoter is operably linked to the DNA encoding heavychain or light chain comprising an anti-IFN-γ antibody of the inventionby removing the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-10); CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad. USA81:659-663); the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-97); herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:144445); promoter and regulatory sequences from themetallothionine gene (Brinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.,75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad.Sci. U.S.A., 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:63946; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399409 (1986); MacDonald, 1987, Hepatology 7:425-515); theinsulin gene control region that is active in pancreatic beta cells(Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene controlregion that is active in lymphoid cells (Grosschedl et al., 1984, Cell38:647-58; Adames et al., 1985, Nature 318:533-38; Alexander et al.,1987, Mol. Cell. Biol, 7:1436-44); the mouse mammary tumor virus controlregion that is active in testicular, breast, lymphoid and mast cells(Leder et al., 1986, Cell 45:485-95); the albumin gene control regionthat is active in liver (Pinkert et al., 1987, Genes and Devel.1:268-76); the alpha-feto-protein gene control region that is active inliver (Krumlauf et al., 1985, Mol. Cell. Biol., 5:1639-48; Hammer etal., 1987, Science 235:53-58); the alpha 1-antitrypsin gene controlregion that is active in liver (Kelsey et al., 1987, Genes and Devel.1:161-71); the beta-globin gene control region that is active in myeloidcells (Mogram et al., 1985, Nature 315:33840; Kollias et al., 1986, Cell46:89-94); the myelin basic protein gene control region that is activein oligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-12); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-86); and the gonadotropicreleasing hormone gene control region that is active in the hypothalamus(Mason et al., 1986, Science 234:1372-78).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising ananti-IFN-γ antibody of the invention by higher eukaryotes. Enhancers arecis-acting elements of DNA, usually about 10-300 by in length, that acton the promoter to increase transcription. Enhancers are relativelyorientation and position independent, having been found at positionsboth 5′ and 3′ to the transcription unit. Several enhancer sequencesavailable from mammalian genes are known (e.g., globin, elastase,albumin, alpha-feto-protein and insulin). Typically, however, anenhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyorna enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer may be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter.

A sequence encoding an appropriate native or heterologous signalsequence (leader sequence or signal peptide) can be incorporated into anexpression vector, to promote extracellular secretion of the antibody.The choice of signal peptide or leader depends on the type of host cellsin which the antibody is to be produced, and a heterologous signalsequence can replace the native signal sequence. Examples of signalpeptides that are functional in mammalian host cells include thefollowing: the signal sequence for interleukin-7 (IL-7) described inU.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptordescribed in Cosman et al. (1984, Nature 312: 768); the interleukin-4receptor signal peptide described in EP Patent No. 0 367 566; the type Iinterleukin-1 receptor signal peptide described in U.S. Pat. No.4,968,607; the type II interleukin-1 receptor signal peptide describedin EP Patent No. 0 460 846; the signal sequence of human IgK (which isMETDTLLLWVLLLWVPGSTG; SEQ ID NO: 52); the signal sequence of humangrowth hormone (which is MATGSRTSLLLAFGLLCLPWLQEGSA; SEQ ID NO: 53); andthe human signal sequences MGSTAILALLLAVLQGVCA (SEQ ID NO: 54) andMETPAQLLFLLLLWLPDTTG (SEQ ID NO: 55), which were encoded by cDNAsencoding the heavy and light chains isolated in Example 3.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chaincomprising an anti-IFN-γ antibody has been inserted into the proper siteof the vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression. The transformationof an expression vector for an anti-IFN-γ antibody into a selected hostcell may be accomplished by well known methods including transfection,infection, calcium phosphate co-precipitation, electroporation,microinjection, lipofection, DEAE-dextran mediated transfection, orother known techniques. The method selected will in part be a functionof the type of host cell to be used. These methods and other suitablemethods are well known to the skilled artisan, and are set forth, forexample, in Sambrook et al., supra.

A host cell, when cultured under appropriate conditions, synthesizes ananti-IFN-γ antibody that can subsequently be collected from the culturemedium (if the host cell secretes it into the medium) or directly fromthe host cell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines may be selected throughdetermining which cell lines have high expression levels andconstitutively produce antibodies with IFN-γ binding properties. Inanother embodiment, a cell line from the B cell lineage that does notmake its own antibody but has a capacity to make and secrete aheterologous antibody can be selected.

Antibodies of the invention are useful for detecting IFN-γ in biologicalsamples and identification of cells or tissues that produce IFN-γprotein. Antibodies of the invention that specifically bind to IFN-γ maybe useful in treatment of IFN-γ mediated diseases. Said antibodies canbe used in binding assays to detect IFN-γ and to inhibit IFN-γ fromforming a complex with IFN-γ receptors. Said antibodies that bind toIFN-γ and block interaction with other binding compounds may havetherapeutic use in modulating IFN-γ mediated diseases. In preferredembodiments, antibodies to IFN-γ may block IFN-γ binding to itsreceptor, which may result in disruption of the IFN-γ induced signaltransduction cascade.

In some embodiments, the invention provides pharmaceutical compositionscomprising a therapeutically effective amount of one or a plurality ofthe antibodies of the invention together with a pharmaceuticallyacceptable diluent, carrier, solubilizer, emulsifier, preservative,and/or adjuvant. Preferably, acceptable formulation materials arenontoxic to recipients at the dosages and concentrations employed. Inpreferred embodiments, pharmaceutical compositions comprising atherapeutically effective amount of anti-IFN-γ antibodies are provided.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed.

In certain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. SeeREMINGTON'S PHARMACEUTICAL SCIENCES, 18^(th) Edition, (A. R. Gennaro,ed.), 1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the invention.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition may be either aqueous or non-aqueous innature. For example, a suitable vehicle or carrier may be water forinjection, physiological saline solution or artificial cerebrospinalfluid, possibly supplemented with other materials common in compositionsfor parenteral administration. Neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In preferredembodiments, pharmaceutical compositions comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may furtherinclude sorbitol or a suitable substitute therefor. In certainembodiments of the invention, anti-IFN-γ antibody compositions may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (REMINGTON'SPHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, the anti-IFN-γantibody product may be formulated as a lyophilizate using appropriateexcipients such as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art.

The formulation components are present preferably in concentrations thatare acceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired anti-IFN-γ antibody in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which the anti-IFN-γ antibody is formulated as asterile, isotonic solution, properly preserved. In certain embodiments,the preparation can involve the formulation of the desired molecule withan agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that may provide controlled or sustained release ofthe product which can be delivered via depot injection. In certainembodiments, hyaluronic acid may also be used, having the effect ofpromoting sustained duration in the circulation. In certain embodiments,implantable drug delivery devices may be used to introduce the desiredantibody molecule.

Pharmaceutical compositions of the invention can be formulated forinhalation. In these embodiments, anti-IFN-γ antibodies areadvantageously formulated as a dry, inhalable powder. In preferredembodiments, anti-IFN-γ antibody inhalation solutions may also beformulated with a propellant for aerosol delivery. In certainembodiments, solutions may be nebulized. Pulmonary administration andformulation methods therefore are further described in InternationalPatent Application No. PCT/US94/001875, which is incorporated byreference and describes pulmonary delivery of chemically modifiedproteins.

It is also contemplated that formulations can be administered orally.Anti-IFN-γ antibodies that are administered in this fashion can beformulated with or without carriers customarily used in the compoundingof solid dosage forms such as tablets and capsules. In certainembodiments, a capsule may be designed to release the active portion ofthe formulation at the point in the gastrointestinal tract whenbioavailability is maximized and pre-systemic degradation is minimized.Additional agents can be included to facilitate absorption of theanti-IFN-γ antibody. Diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders may also be employed.

A pharmaceutical composition of the invention is preferably provided tocomprise an effective quantity of one or a plurality of anti-IFN-γantibodies in a mixture with non-toxic excipients that are suitable forthe manufacture of tablets. By dissolving the tablets in sterile water,or another appropriate vehicle, solutions may be prepared in unit-doseform. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving anti-IFN-γ antibodies insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, International Patent Application No. PCT/US93/00829, whichis incorporated by reference and describes controlled release of porouspolymeric microparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g. films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-556),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent Application PublicationNo. EP 133,988). Sustained release compositions may also includeliposomes that can be prepared by any of several methods known in theart. See e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA82:3688-3692; European Patent Application Publication Nos. EP 036,676;EP 088,046 and EP 143,949, incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration.

The invention also provides kits for producing a single-doseadministration unit. The kits of the invention may each contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments of this invention, kitscontaining single and multi-chambered pre-filled syringes (e.g., liquidsyringes and lyosyringes) are provided.

The therapeutically effective amount of an anti-IFN-γantibody-containing pharmaceutical composition to be employed willdepend, for example, upon the therapeutic context and objectives. Oneskilled in the art will appreciate that the appropriate dosage levelsfor treatment will vary depending, in part, upon the molecule delivered,the indication for which the anti-IFN-γ antibody is being used, theroute of administration, and the size (body weight, body surface ororgan size) and/or condition (the age and general health) of thepatient. In certain embodiments, the clinician may titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 μg/kg to up to about30 mg/kg or more, depending on the factors mentioned above. In preferredembodiments, the dosage may range from 0.1 μg/kg up to about 30 mg/kg,optionally from 1 μg/kg up to about 30 mg/kg or from 10 μg/kg up toabout 5 mg/kg.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular anti-IFN-γ antibody in the formulation used. Typically, aclinician administers the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data. In certain embodiments, the antibodiesof the invention can be administered to patients throughout an extendedtime period. Chronic administration of an antibody of the inventionminimizes the adverse immune or allergic response commonly associatedwith antibodies that are raised against a human antigen in a non-humananimal, for example, a non-fully human antibody or non-human antibodyproduced in a non-human species.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; by sustained release systems or byimplantation devices. In certain embodiments, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

The composition also may be administered locally via implantation of amembrane, sponge or another appropriate material onto which the desiredmolecule has been absorbed or encapsulated. In certain embodiments,where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of the desired molecule maybe via diffusion, timed-release bolus, or continuous administration.

It also may be desirable to use anti-IFN-γ antibody pharmaceuticalcompositions according to the invention ex vivo. In such instances,cells, tissues or organs that have been removed from the patient areexposed to anti-IFN-γ antibody pharmaceutical compositions after whichthe cells, tissues and/or organs are subsequently implanted back intothe patient.

In particular, anti-IFN-γ antibodies can be delivered by implantingcertain cells that have been genetically engineered, using methods suchas those described herein, to express and secrete the polypeptide. Incertain embodiments, such cells may be animal or human cells, and may beautologous, heterologous, or xenogeneic. In certain embodiments, thecells may be immortalized. In other embodiments, in order to decreasethe chance of an immunological response, the cells may be encapsulatedto avoid infiltration of surrounding tissues. In further embodiments,the encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting the invention.

Example 1 Generation of Human IFNγ Protein from CHO Cells

The full-length human IFN-γ cDNA was amplified by PCR (under standardconditions) using human spleen Marathon-Ready cDNA (Clontech) as atemplate. The sequence was subcloned into the pDSR.alpha.2 plasmid.DH10B (Escherichia coli) cells were transformed with the pDSR.alpha.2plasmid. DNA was prepared using standard techniques, and CHO cells weretransfected by the calcium phosphate method (Speciality Media, Inc.). Ahigh-expressing cell line clone was used to generate serum-freeconditioned media.

CHO cell conditioned media containing human IFN-γ (hu-IFN-γ wasconcentrated, dialyzed, and purified through several chromatographysteps. The first step was Q-HP (Pharmacia) chromatography using astandard NaCl gradient to separate highly glycosylated fromunglycosylated hu-IFN-γ forms. The Q-HP pool was further purifiedthrough wheat germ agglutinin chromatography (EY Laboratories). Thepurified material was separated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and analyzed by Coomassie-blue andsilver-staining. The purified material was greater than 95% pure asdetermined by both Coomassie-blue and silver-stained SDS-PAGE. Thematerial was also assayed by the gel-clot method (Limulus AmebocyteLysate), indicating a low endotoxin level. The identity of hu-IFN-γ wasconfirmed by Western blotting using anti-AF-285 NA antibody from R & DSystems. The final protein concentration was determined from absorbance(A₂₈₀) using the extinction coefficient method, where A₂₈₀reading/extinction coefficient=concentration in g/L (extinctioncoefficient=0.66).

Example 2 Production of Human Monoclonal Antibodies Against IFN-γ

Transgenic HuMab Mice

Fully human monoclonal antibodies to IFN-γ were prepared using HCo7,HCo12, and HCo7+HCo12 strains of transgenic mice, each of whichexpressed human antibody genes. In each of these strains, the endogenousmouse kappa light chain gene had been homozygously disrupted asdescribed in Chen et al. (1993, EMBO J. 12:811-820), and the endogenousmouse heavy chain gene had been homozygously disrupted as described inExample 1 of International Patent Application Publication No. WO01/09187 (incorporated by reference). Each strain carried a human kappalight chain transgene, KCo5, as described in Fishwild et al. (1996,Nature Biotechnology 14:845-851). The HCo7 strain carries the HCo7 humanheavy chain transgene as described in U.S. Pat. Nos. 5,545,806,5,625,825, and 5,545,807 (incorporated by reference). The HCo12 straincarried the HCo12 human heavy chain transgene as described in Example 2of International Patent Application Publication No. WO 01/09187(incorporated by reference). The HCo7+HCo12 strain carried both the HCo7and the HCo12 heavy chain transgenes and was hemizygous for eachtransgene. All of these strains are referred to herein as HuMab mice.

HuMab Immunizations:

To generate fully human monoclonal antibodies to IFN-γ, HuMab mice wereimmunized with purified recombinant human IFN-γ derived from E. coli orCHO cells as antigen. General immunization schemes for HuMab mice aredescribed in Lonberg et al. (1994, Nature 368:856-859; Fishwild et al.,supra., and International Patent Application Publication No. WO98/24884, the teachings of each of which are incorporated by reference).Mice were 6-16 weeks of age upon the first infusion of antigen. Apurified recombinant preparation (25-100 μg) of IFN-γ antigen (e.g.,purified from transfected E. coli or CHO cells expressing IFN-γ) wasused to immunize the HuMab mice intraperitoneally (IP) or subcutaneously(Sc).

Immunizations of HuMab transgenic mice were achieved using antigen incomplete Freund's adjuvant and two injections, followed by 2-4 weeks IPimmunization (up to a total of 9 immunizations) with the antigen inincomplete Freund's adjuvant. Several dozen mice were immunized for eachantigen (human IFN-γ produced in either E. coli or CHO cells). A totalof 91 mice of the HCo7, HCo12, and HCo7+HCo12 strains were immunizedwith IFN-γ. The immune response was monitored by retroorbital bleeds.

To select HuMab mice producing antibodies that bound IFN-γ, sera fromimmunized mice was tested by ELISA as described by Fishwild et al.supra. Briefly, microtiter plates were coated with purified recombinantIFN-γ from CHO cells or E. coli at 1-2 μL/mL in PBS and 50 μL/wellincubated at 4° C. overnight, then blocked with 200 μL/well of 5%chicken serum in PBS/Tween (0.05%). Dilutions of plasma fromIFN-γ-immunized mice were added to each well and incubated for 1-2 hoursat ambient temperature. The plates were washed with PBS/Tween and thenincubated with a goat-anti-human IgG Fc-specific polyclonal reagentconjugated to horseradish peroxidase (HRP) for 1 hour at roomtemperature. Plates were washed with PBS/Tween and incubated with a goatanti-human IgG Fc-specific polyclonal reagent conjugated to horseradishperoxidase (HRP) for 1 hour at room temperature. After washing, theplates were developed with ABTS substrate (Sigma Chemical Co., St.Louis, Mo., Catalog No. A-1888, 0.22 mg/mL) and analyzedspectrophotometrically by determining optical density (OD) atwavelengths from 415-495 nm. Mice with sufficient titers of anti-IFN-γhuman immunoglobulin were used to produce monoclonal antibodies asdescribed below.

Generation of Hybridomas Producing Human Monoclonal Antibodies to IFN-γ.

Mice were prepared for monoclonal antibody production by boosting withantigen intravenously 2 days before sacrifice, and spleens were removedthereafter. The mouse splenocytes were isolated from the HuMab mice andfused with PEG to a mouse myeloma cell line using standard protocols.Typically, 10-20 fusions for each antigen were performed.

Briefly, single cell suspensions of splenic lymphocytes from immunizedmice were fused to one-fourth the number of P3×63-Ag8.653 nonsecretingmouse myeloma cells (ATCC, Accession No. CRL 1580) with 50% PEG (Sigma).Cells were plated at approximately 1×10⁵/well in flat bottom microtiterplates, followed by about a two week incubation in selective mediumcontaining 10% fetal bovine serum, 10% P388D1-(ATCC, Accession No. CRLTIB-63) conditioned medium, 3-5% ORIGEN® Hybridoma Cloning Factor(IGEN), a partially purified hybridoma growth medium supplement derivedfrom medium used to culture a murine macrophage-like cell line, in DMEM(Mediatech, Catalog No. CRL 10013, with high glucose, L-glutamine, andsodium pyruvate) plus 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/mLgentamycin, and 1×HAT (Sigma, Catalog No. CRL P-7185). After 1-2 weeks,cells were cultured in medium in which the HAT was replaced with HT.

The resulting hybridomas were screened for the production ofantigen-specific antibodies. Individual wells were screened by ELISA(described above) for human anti-IFN-γ monoclonal IgG antibodies. Onceextensive hybridoma growth occurred, medium was monitored, usually after10-14 days. Antibody secreting hybridomas were replated, screened againand, if still positive for human IgG, anti-IFN-γ monoclonal antibodieswere subcloned at least twice by limiting dilution. The stable subcloneswere then cultured in vitro to generate small amounts of antibody intissue culture medium for purification and characterization.

Selection of Human Monoclonal Antibodies that Bind to IFN-γ

An ELISA assay as described above was used to screen for hybridomas thatshowed positive reactivity with IFN-γ immunogen. Hybridomas secreting amonoclonal antibody that bound with high avidity to IFN-γ were subclonedand further characterized. One clone from each hybridoma, which retainedthe reactivity of parent cells (as determined by ELISA), was chosen formaking a 5-10 vial cell bank stored in liquid nitrogen.

An isotype-specific ELISA was performed to determine the isotype of themonoclonal antibodies produced as disclosed herein. In theseexperiments, microtiter plate wells were coated with 50 μL/well of asolution of 1 μg/mL of mouse anti-human kappa light chain in PBS andincubated at 4° C. overnight. After blocking with 5% chicken serum, theplates were reacted with supernatant from each tested monoclonalantibody and a purified isotype control. Plates were incubated atambient temperature for 1-2 hours. The wells were then reacted witheither human IgG1 or IgG3-specific horseradish peroxidase-conjugatedgoat anti-human polyclonal antisera, and plates were developed andanalyzed as described above.

Monoclonal antibodies purified from the hybridoma supernatants thatshowed significant binding to IFN-γ as detected by ELISA were furthertested for biological activity using a variety of bioassays as describedbelow. The antibodies selected were designated 1119, 1121, 1118*, 1121*,and 1118.

Example 3

Cloning the Anti-IFN-γ Antibody Heavy and Light Chains

The hybridomas expressing IFN-γ binding monoclonal antibodies 1119,1121, 1118*, 1121*, and 1118 identified in Example 2 above were used assources to isolate total RNA using TRIzol® reagent (Invitrogen), amonophasic solution of phenol and guanidine isothiocyanate suitable forisolating total RNA, DNA, and protein. First strand cDNA was synthesizedusing a random primer with an extension adapter (5′-GGC CGG ATA GGC CTCCAN NNN NNT-3′) (SEQ ID NO:23) and a 5′ RACE (rapid amplification ofcDNA ends) preparative assay was performed using the GENERACER™ Kit(Invitrogen), a kit for rapid amplification of cDNA ends (RACE) withimproved efficiency, according to instructions from the manufacturer.For preparing complete light chain-encoding cDNA, the forward primer wasthe GENERACER™ nested primer, and the reverse primer was 5′-GGG GTC AGGCTG GAA CTG AGG-3′ (SEQ ID NO:24). The reverse primer was designed torecognize a conserved region of the cDNA sequence found in the 3′untranslated region of human kappa chains. For preparing cDNA encodingthe variable region of the heavy chains, the forward primer was theGENERACER™ nested primer and the reverse primer was 5′-TGA GGA CGC TGACCA CAC G-3′ (SEQ ID NO:25), which was designed to recognize a conservedregion in the coding sequence in the Fc region of human IgG chains. RACEproducts were cloned into pCR4-TOPO (Invitrogen), and the sequences weredetermined. Consensus sequences were used to design primers forfull-length antibody chain PCR amplification.

For preparing cDNA encoding anti-IFN-γ kappa light chain, the 5′ PCRprimer encoded the amino terminus of the signal sequence, an XbaIrestriction enzyme site, and an optimized Kozak sequence (5′-ACA ACA AAGCTT CTA GAC CAC CAT GGA AAC CCC AGC TCA GCT TCT CTT-3′; SEQ ID NO:26).The 3′ primer encoded the carboxyl terminus and termination codon, aswell as a SalI restriction site (5′-CTT GTC GAC TCA ACA CTC TCC CCT GTTGAA GCT-3′; SEQ ID NO:27). The resulting PCR product fragment waspurified, digested with XbaI and SalI, and then gel isolated and ligatedinto the mammalian expression vector pDSR.alpha.9 (see InternationalApplication, Publication No. WO 90/14363, which is herein incorporatedby reference for any purpose).

For preparing cDNA encoding anti-IFN-γ heavy chain the 5′ PCR primerencoded the amino terminus of the signal sequence, an XbaI restrictionenzyme site, and an optimized Kozak sequence (5′-CAG CAG AAG CTT CTA GACCAC CAT GGG GTC AAC CGC CAT CCT CG-3′; SEQ ID NO:28). The 3′ primerencoded the carboxyl end of the variable region, including a naturallyoccurring sense strand BsmBI site (5′-CTT GGT GGA GGC ACT AGA GAC GGTGAC CAG GGT GCC ACG GCC-3′; SEQ ID NO:29). The resulting product waspurified, digested with XbaI and BsmBI, gel isolated and ligated intothe pDSR.alpha.19 vector containing the human IgG1 constant region.

Example 4 Expression of Anti-IFN-γ Antibodies in Chinese Hamster Ovary(CHO) Cells

Stable expression of the 1119 anti-IFN-γ mAb was achieved byco-transfection of 1119-heavy chain/pDSR.alpha.19 and 1119-kappachain/pDSR.alpha.19 plasmids into dihydrofolate reductase deficient(DHFR⁻), serum-free adapted Chinese hamster ovary (CHO) cells using acalcium phosphate method. Transfected cells were selected in mediumcontaining dialyzed serum but not containing hypoxanthine-thymidine toensure the growth of cells expressing the DHFR enzyme. Transfectedclones were screened using assays such as ELISA in order to detect theexpression of 1119 anti-IFN-γ mAb in the conditioned medium. The1119-expressing cell lines were subjected to methotrexate amplification.The highest expressing clones upon amplification were selected forsingle cell cloning and creation of cell banks

Any recombinant anti-IFN-γ antibody of the invention can be generated inChinese hamster ovary cells deficient in DHFR using the same protocol asdescribed above for the 1119 MAb. The DNA sequences encoding thecomplete heavy chain or light chain of each anti-IFN-γ antibody of theinvention are cloned into expression vectors. CHO cells deficient inDHFR are co-transfected with an expression vector capable of expressinga complete heavy chain and an expression vector expressing the completelight chain of the appropriate anti-IFN-γ antibody. For example, togenerate the 1118 antibody, cells are co-transfected with a vectorcapable of expressing a complete heavy chain comprising the amino acidsequence as set forth in SEQ ID NO: 19 and a vector capable ofexpressing a complete light chain comprising the amino acid sequence setforth in SEQ ID NO: 20. To generate the 1121 antibody, cells areco-transfected with a vector capable of expressing a complete heavychain comprising the amino acid sequence as set forth in SEQ ID NO: 21and a vector capable of expressing a complete light chain comprising theamino acid sequence set forth in SEQ ID NO: 22. To generate the 1118*antibody, cells are co-transfected with a vector capable of expressing acomplete heavy chain comprising the amino acid sequence as set forth inSEQ ID NO: 32 and a vector capable of expressing a complete light chaincomprising the amino acid sequence set forth in SEQ ID NO: 20. Togenerate the 1121* antibody, cells are co-transfected with a vectorcapable of expressing a complete heavy chain comprising the amino acidsequence as set forth in SEQ ID NO: 21 and a vector capable ofexpressing a complete light chain comprising the amino acid sequence setforth in SEQ ID NO: 33. Table 3 summarizes the complete heavy andcomplete light chains for the various IFN-γ antibodies.

TABLE 3 Heavy Chain Variable Region + Heavy Chain Constant CompleteHeavy Antibody Region Chain 1119 SEQ ID NO: 6 + SEQ ID NO: 2 SEQ ID NO:17 1118 SEQ ID NO: 10 + SEQ ID NO: 2 SEQ ID NO: 19 1121 SEQ ID NO: 14 +SEQ ID NO: 2 SEQ ID NO: 21 1121* SEQ ID NO: 14 + SEQ ID NO: 2 SEQ ID NO:21 1118* SEQ ID NO: 30 + SEQ ID NO: 2 SEQ ID NO: 32 Light Chain VariableRegion + Light Chain Constant Complete Light Antibody Region Chain 1119SEQ ID NO: 8 + SEQ ID NO: 4 SEQ ID NO: 18 1118 SEQ ID NO: 12 + SEQ IDNO: 4 SEQ ID NO: 20 1121 SEQ ID NO: 16 + SEQ ID NO: 4 SEQ ID NO: 221121* SEQ ID NO: 31 + SEQ ID NO: 4 SEQ ID NO: 33 1118* SEQ ID NO: 12 +SEQ ID NO: 4 SEQ ID NO: 20

Example 5 Production of Anti-IFN-γ Antibody

The 1119 antibody was produced by expression in a clonal line of CHOcells that expressed it. For the production run, cells from a singlevial were thawed into serum-free cell culture media. The cells weregrown initially in a 250 mL shake flask, then in spinner flasks, andfinally in stainless steel reactors of increasing scale up to a 2000 Lbioreactor. Production was carried out in a 2000 L bioreactor using afed batch culture, in which a nutrient feed containing concentratedmedia components is added to maintain cell growth and culture viability.Production lasted for approximately two weeks, during which time the1119 antibody was constitutively produced by the cells and secreted intothe cell culture medium.

The production reactor was controlled at a predetermined pH,temperature, and dissolved oxygen level. The pH was controlled by carbondioxide gas and sodium carbonate addition. Dissolved oxygen wascontrolled by air, nitrogen, and oxygen gas flows.

At the end of production, the cell broth was fed into a disk stackcentrifuge, and the culture supernatant was separated from the cells.The concentrate is further clarified through a depth filter followed bya 0.2 μm filter. The clarified conditioned media was then concentratedby tangential flow ultrafiltration. The conditioned media wasconcentrated 15- to 30-fold. The resulting concentrated conditionedmedium was then processed to purify the antibody it contains, but it maybe frozen for purification at a later date. Any of the other antibodiesdescribed herein could be produced in a similar fashion.

Example 6

Characterizing the Activity of Anti-IFN-γ Antibodies

Since IFN-γ has a large number of biological effects, several differentbioassays were used to compare the potency of various IFN-γ antibodies.The A549 assay described below was used for the primary screen withcandidates selected for further analysis based on their performance inthe assay. Selected candidates included the 1119, 1118, and 1121antibodies.

A549 Bioassay

One of the established properties of IFN-γ is its anti-proliferativeeffect on a variety of cell populations. See e.g. Aune and Pogue, 1989,J. Clin. Invest. 84:863-75. The human lung cell line A549 has been usedfrequently in publications describing the bioactivity of IFN-γ. See e.g.Aune and Pogue, supra; Hill et al., 1993, Immunology 79:236-40. Ingeneral, the activity of an inhibitor is tested at a concentration of astimulating substance that falls within a part of the dose-responsecurve where a small change in dose will result in a change in response.One of skill in the art will realize that if an excessive dose of thestimulating substance is used, a very large dose of an inhibitor may berequired to observe a change in response. Commonly used concentrationsfor a stimulating substance are EC₈₀ and EC₉₀ (the concentrations atwhich 80% or 90%, respectively, of the maximum response is achieved).

An IFN-γ dose-response curve was generated to determine the EC₉₀ for thelung epithelial carcinoma cell line A549 (−30 μM). In subsequentexperiments, different concentrations of purified antibodies were mixedwith a fixed dose of IFN-γ (30 μM), and the ability of the antibodies toinhibit the biological activity of the anti-proliferative effect ofIFN-γ was determined. The assay was performed for 5 days, andproliferation was measured by determining fluorescence generated by thereduction of ALAMARBLUE™ (AccuMed International, Inc., Chicago, Ill.), adye used to indicate cell growth, by metabolically active, i.e.,proliferating, cells. See e.g., de Fries and Mitsuhashi, 1995, J. Clin.Lab. Analysis 9(2):89-95; Ahmed et al., 1994, J. Immunol. Methods170(2):211-24.

As shown in FIG. 9, the 1119 antibody was the most potent antibody withan IC₅₀ (concentration at which 50% inhibition of the effect of IFN-γwas achieved) of 14 μM, followed by 1121 (46 μM), and 1118 (97 μM).

HLA DR Bioassay

Another established property of IFN-γ is its ability to upregulate theexpression of MHC Class I and Class II genes in a variety of cell types.This activity may be particularly relevant to lupus nephritis (Yokoyamaet al., 1992, Kidney Int. 42:755-63). The THP-1 human monocytic cellline has been used frequently in publications describing thisbioactivity of IFN-γ. An IFN-γ dose-response curve was generated todetermine the EC₈₀ for the particular THP-1 cell line used in thisexperiment (−21 μM). In subsequent experiments, different concentrationsof purified antibodies were mixed with a fixed dose of IFN-γ (21 μM) andthe ability of the antibodies to neutralize or inhibit the IFN-γ-inducedupregulation of HLA DR expression on the cell surface was determined.The assay was performed for 24 hours, and the measured endpoint was meanfluorescence intensity as determined by FACS analysis to detect bindingof a FITC-labeled anti-HLA DR antibody to the cells.

As shown in FIG. 10, the 1119 antibody was the most potent antibody withan IC₅₀ of 14 μM, followed by 1121 (60 μM), and 1118 (86 μM).

Whole Blood Bioassay

A human whole blood assay was developed based on published observationsthat IFN-γ upregulates the production of the IP-10 chemokine in severaldifferent cell lines. This activity may be particularly relevant tolupus nephritis (Narumi et al., 2000, Cytokine 12:1561-1565). Wholeblood from a number of normal human donors was tested for the ability ofIFN-γ to increase IP-10 production. An IFN-γ dose-response curve wasgenerated to determine the EC_(so) for individual donors. As expected,some variation was observed between donors. In general, donors were usedthat appeared to reproducibly display an EC₅₀ of 50-100 μM. Whole bloodwas mixed with a fixed concentration of IFN-γ and differentconcentrations of antibodies, incubated for 18.5 hours and then IP-10levels determined by ELISA. Representative results from a whole bloodassay for two different donors are shown in FIG. 11. The IC₅₀s fromthese two donors were 17 and 14 μM. To date, one donor has beenidentified with spontaneously elevated IP-10 levels in the whole bloodassay without need for the addition of exogenous IFN-γ. The anti-IFN-γantibodies were capable of blocking this spontaneous production of IP-10presumably by blocking the endogenously produced IFN-γ.

Biochemical Assays

Binding kinetics for several of the antibodies to IFN-γ were measured byBIAcore analysis. Initial results suggested that the antibodies hadoff-rates that approached the limitations for reliable measurements onthe BIACORE™ (Pharmacia Biosensor AB Corporation, Uppsala, Sweden), anapparatus that uses surface plasmon resonance to measure binding betweenmolecules. Accordingly, an equilibrium-binding assay was developed andused. A fixed amount of antibody was incubated with variousconcentrations of IFN-γ for greater than 5 hours in order to reachequilibrium and then contacted with IFN-γ coupled beads for a very brieftime, and the amount of free antibody that bound to the beads wasmeasured in a KINEXA™ machine (Sapidyne Instruments Inc., Boise, Id.), afluorescence based immunoassay instrument. The lowest equilibriumdissociation constant obtained, ˜24 μM, was with the 1119 antibody.

Example 7

Species Cross-Reactivity

The antibodies described above were tested for their ability toneutralize or inhibit recombinant IFN-γ proteins from several differentspecies. The mouse IFN-γ protein was purchased commercially, while thehuman, cynomolgus monkey and chimpanzee IFN-γ proteins were cloned andexpressed in conventional mammalian expression systems such as human 293cells. The human, cynomolgus, and chimpanzee IFN-γ proteins were allactive in the previously described A549 assay while the mouse proteinwas not active in this assay. The mouse protein was active in a RAW264.7 cell-line based assay, which was essentially identical to the A549assay described previously except for the substitution of the mouse cellline. RAW 264.7 is a mouse monocytic macrophage cell line and can beobtained from, for example, the American Type Culture Collection. Asshown in Table 4, all three antibodies were able to neutralize human andchimpanzee IFN-γ, while none of the three were able to neutralize orinhibit the biological activity of IFN-γ from either cynomolgus ormouse.

TABLE 4 Antibody Human Chimp. Cyno. Mouse 1118 Yes Yes No No 1119 YesYes No No 1121 Yes Yes No No

Example 8 Identification of an Epitope for Anti-IFN-γ Antibodies

A comparison of the amino acid sequences of mature human and cynomolgusIFN-γ indicated that there were nine amino acid differences between themat positions 19, 20, 31, 34, 65, 77, 103, 110, and 126 in the humanIFN-γ sequence. Human and chimpanzee IFN-γ sequences are disclosed inThakur and Landolfi (1999), Molecular Immunology 36: 1107-15, Thecynomolgus monkey IFN-γ sequence is disclosed in Tatsumi and Sata(1997), Int. Arch. Allergy Immunol. 114(3): 229-36; and the murine IFN-γsequence is disclosed in, e.g., National Center for BiotechnologyInformation (NCBI) Accession No. NP 032363. Site-directed mutagenesisusing a commercially available kit was used to substitute individuallyeach of the divergent human amino acids within the human IFN-γ with thecorresponding amino acid from the cynomolgus protein. Each substitutedIFN-γ was named “huIFN-γ” followed by the symbol for the amino acid usedto replace the amino acid present in the human IFN-γ sequence and theposition in the mature human IFN-γ sequence at which the substitutionoccurred. For example, “huIFNyDl9” represents a version of IFN-γidentical to human IFN-γ except at position 19, where an aspartic acidreplaces the histidine normally occupying this position. Similarexperiments were done starting with the cynomolgus monkey IFN-γ andsubstituting each divergent amino acid with the amino acid present inhuman IFN-γ. For example, “cynolFNyD 103” represents a version of IFN-γidentical to cynomolgus monkey IFN-γ except at position 103, where aleucine replaces the serine normally occupying this position. See Tables5 and 6. These mutant proteins were expressed in a conventionalmammalian expression system such as the human 293 cells. All the mutantIFN-γ proteins retained activity as determined in the A549 assay. The1119 antibody was tested for its ability to neutralize or inhibit thebiological activity of the various mutant IFN-γ proteins, as determinedusing an A549 bioassay. The ability of the 1119 antibody to neutralizeor inhibit human IFN-γ anti-proliferative activity was determined bymeasuring fluorescence as described above, and this was used as abaseline for comparing the ability of the 1119 antibody to neutralizethe activity of each of the variant forms of IFN-γ. For the constructsthat started with human IFN-γ, inhibition of IFN-γ activity was measuredas a percentage, where maximal level of fluorescence observed in thepresence of the human IFN-γ and the 1119 antibody (due to reduction ofALAMARBLUE™ by proliferating cells) was set to 100%, and the maximallevels measured in the presence of each of the altered forms of IFN-γplus the 1119 antibody were compared to this. Alternatively, for theconstructs that started with cynomolgus monkey IFN-γ, inhibition wasscored qualitatively based on the observed fluorescence.

As summarized in Table 5, the 1119 antibody was able to neutralize orinhibit the biological activity of human IFN-γ and all the substitutionmutants of human IFN-γ except for huIFNγD19 and huIFNγP20. As in theprevious example, the cynomolgus IFN-γ protein was also not inhibited bythe 1119 antibody. This analysis indicates that residues 19 and 20 areparticularly important for the interaction between the 1119 antibody andIFN-γ and may serve as points of contact between the 1119 antibody andhuman IFN-γ.

TABLE 5 IFN-γ % Neutralization by 1119 Human IFN-γ 100 Cynomolgus IFN-γ0 huIFN-γD19 0 huIFN-γP20 0 huIFN-γD31 103 huIFN-γR34 109 huIFN-γR65 109huIFN-γI77 108 huIFN-γS103 102 huIFN-γV110 103 huIFN-γI126 109

Table 6 shows that an altered version of the cynomolgus monkey IFN-γcomprising substitutions making the cynomolgus IFNγ sequence match thehuman sequence at positions 19 and 20 was not neutralized by the 1119antibody. However, versions of the cynomolgus monkey IFNγ having humansequence substitutions at positions 19, 20, and 65, or 19, 20, and 103were neutralized by the 1119 antibody, as were versions containingsubstitutions in addition to either of these.

TABLE 6 Construct Neutralization by 1119 human IFNγ yes cyno IFNγ nocyno IFNγH19/S20 no cyno IFNγS65 no cyno IFNγL103 no cyno IFNγS65/L103no cyno IFNγH19/S20/S65 yes cyno IFNγH19/S20/L103 yes cynoIFNγH19/S20/L103/I110 yes cyno yes IFNγH19/S20/S65/L103/I110

Example 9 Biological Activity of Anti-IFN-γ Antibody Upon Administrationto Chimpanzees

The 1119 antibody was administered to two chimpanzees at a dose of 20mg/kg every week for three weeks. Blood was drawn from the chimpanzeeseither two (−2) or one week (−1) before administration of antibody, andfurther blood was drawn at 2, 8, 15, 29, and 36 days afteradministration of the first dose of antibody. A chimpanzee whole bloodassay was performed on the drawn blood using essentially the same methodused in the human whole blood assay described in Example 6, the keydistinction being that the antibody was not added to the whole bloodexogenously but was administered previously in vivo to the chimpanzees.IFN-γ was added to the blood at various concentrations (0.01 ng/ml, 3.9ng/ml, or 1 ug/ml), the blood was incubated at 37° C. for 20-24 hours,and then IP-10 production was measured by bead-based ELISA.

As can be seen in FIGS. 14 and 15, blood drawn from animals prior toantibody dosing responded to IFN-γ with a concentration dependentincrease in IP-10 production. In contrast, blood drawn after antibodyadministration did not respond to any of the concentrations of IFN-γtested with an increase in IP-10 production. These data indicate thatthe antibody retained the ability to neutralize IFN-γ, even afteradministration in vivo. Further, the amount of IFN-γ added to thesecultures greatly exceeded the endogenous levels of IFN-γ in thechimpanzees, suggesting that the administered antibody would be capableof neutralizing endogenous IFN-γ in vivo.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims. All references citedherein are incorporated by reference in their entirety.

1. An isolated nucleic acid that encodes a human IgG antibody that bindsto a human Interferon gamma, wherein the antibody comprises (a) a heavychain CDR1 comprising the amino acid sequence of SEQ ID NO:34, a heavychain CDR2 comprising the amino acid sequence of SEQ ID NO:35, and aheavy chain CDR3 of comprising the amino acid sequence SEQ ID NO:36, orSEQ ID NO:37; and/or (b) a light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 38, SEQ ID NO:39, or SEQ ID NO:40; a light chainCDR2 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:42;and a light chain CDR3 comprising the amino acid sequence of SEQ IDNO:43 or SEQ ID NO:44.
 2. The nucleic acid of claim 1, wherein theantibody comprises the heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO:34, the heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO:35, the heavy chain CDR3 comprising the amino acidsequence of SEQ ID NO:37, the light chain CDR1 comprising the amino acidsequence of SEQ ID NO:39, the light chain CDR2 comprising the amino acidsequence of SEQ ID NO:41, and the light chain CDR3 comprising the aminoacid sequence of SEQ ID NO:43.
 3. The nucleic acid of claim 1, whereinthe antibody comprises the heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO:34, the heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO:35, the heavy chain CDR3 comprising the amino acidsequence of SEQ ID NO:36, the light chain CDR1 comprising the amino acidsequence of SEQ ID NO:38, the light chain CDR2 comprising the amino acidsequence of SEQ ID NO:41, and the light chain CDR3 comprising the aminoacid sequence of SEQ ID NO:43.
 4. The nucleic acid of claim 1, whereinthe antibody comprises the heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO:34, the heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO:35, the heavy chain CDR3 comprising the amino acidsequence of SEQ ID NO:37, the light chain CDR1 comprising the amino acidsequence of SEQ ID NO:38, the light chain CDR2 comprising the amino acidsequence of SEQ ID NO:41, and the light chain CDR3 comprising the aminoacid sequence of SEQ ID NO:43.
 5. The nucleic acid of claim 1, whereinthe antibody comprises the heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO:34, the heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO:35, the heavy chain CDR3 comprising the amino acidsequence of SEQ ID NO:37, the light chain CDR1 comprising the amino acidsequence of SEQ ID NO:40, the light chain CDR2 comprising the amino acidsequence of SEQ ID NO:42, and the light chain CDR3 comprising the aminoacid sequence of SEQ ID NO:44.
 6. A host cell comprising the nucleicacid of claim
 1. 7. A method of making an antibody comprising culturingthe host cell of claim 6 under conditions such that it expresses theantibody and collecting the antibody from the culture medium or the hostcell.
 8. A host cell comprising the nucleic acid of claim
 3. 9. A methodof making an antibody comprising culturing the host cell of claim 8under conditions such that it expresses the antibody and collecting theantibody from the culture medium or the host cell.
 10. An isolatednucleic acid which encodes a human IgG antibody that binds to a humanInterferon gamma, wherein the antibody comprises the amino acid sequenceof: SEQ ID NO:6 and/or SEQ ID NO:8; SEQ ID NO:10 and/or SEQ ID NO:12;SEQ ID NO:14 and/or SEQ ID NO:16; SEQ ID NO:30 and/or SEQ ID NO:12; orSEQ ID NO:14 and/or SEQ ID NO:31.
 11. The nucleic acid of claim 10,wherein the antibody comprises the amino acid sequence of SEQ ID NO:8,SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:31.
 12. The nucleic acid ofclaim 11, wherein the antibody comprises an amino acid sequence at least95% identical to SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, or SEQ IDNO:30.
 13. The nucleic acid of claim 10, wherein the antibody comprisesthe amino acid sequence of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, orSEQ ID NO:30.
 14. The nucleic acid of claim 13, wherein the antibodycomprises an amino acid sequence at least 95% identical to SEQ ID NO:8,SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:31.
 15. The nucleic acidsequence of claim 14, wherein the antibody comprises the amino acidsequence of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:31.16. The nucleic acid of claim 15, wherein the antibody comprises theamino acid sequences of SEQ ID NO:6 and SEQ ID NO:8.
 17. A host cellcomprising the nucleic acid of claim
 10. 18. A method of making anantibody comprising culturing the host cell of claim 17 under conditionssuch that it expresses the antibody and collecting the antibody from theculture medium or the host cell.
 19. A host cell comprising the nucleicacid of claim
 13. 20. A method of making an antibody comprisingculturing the host cell of claim 19 under conditions such that itexpresses the antibody and collecting the antibody from the culturemedium or the host cell.
 21. A host cell comprising the nucleic acid ofclaim
 16. 22. A method of making an antibody comprising culturing thehost cell of claim 21 under conditions such that it expresses theantibody and collecting the antibody from the culture medium or the hostcell.
 23. An isolated nucleic acid that encodes a human antibody thatbinds to a human Interferon gamma, wherein the nucleic acid comprisesthe nucleotide sequence of: SEQ ID NO:5 and/or SEQ ID NO:7; SEQ ID NO:9and/or SEQ ID NO:11; SEQ ID NO:13 and/or SEQ ID NO:15; SEQ ID NO:56and/or SEQ ID NO:11; or SEQ ID NO:13 and/or SEQ ID NO:57.
 24. Thenucleic acid of claim 23 comprising SEQ ID NO:5, SEQ ID NO:9, SEQ IDNO:13, or SEQ ID NO:56.
 25. The nucleic acid of claim 23 comprising SEQID NO:7, SEQ ID NO:11, SEQ ID NO:15, or SEQ ID NO:57
 26. The nucleicacid of claim 23 comprising SEQ ID NO:5 and SEQ ID NO:7.
 27. A host cellcomprising the nucleic acid of claim
 23. 28. A method of making anantibody comprising culturing the host cell of claim 27 under conditionssuch that it expresses the antibody and collecting the antibody from theculture medium or the host cell.
 29. A host cell comprising the nucleicacid of claim
 26. 30. A method of making an antibody comprisingculturing the host cell of claim 29 under conditions such that itexpresses the antibody and collecting the antibody from the culturemedium or the host cell.